Genes which influence pichia proteolytic activity, and uses therefor

ABSTRACT

The isolation and characterization of genes involved in proteolytic processing in species of the genus Pichia is described. The availability of such genes has enabled the generation of strains of Pichia which are deficient in proteolytic activity, which strains are useful as hosts for the expression of proteolytically sensitive recombinant products. The isolation and characterization of additional genes from species of the genus Pichia is also described, as well as uses therefore.

This is a continuation of U.S. application Ser. No. 08/245,756, filedMay 16, 1994, now U.S. Pat. No. 5,541,112, which is a divisional of U.S.application Ser. No. 08/088,633, filed Jul. 6, 1993, now U.S. Pat. No.5,324,660, which in turn is a continuation of application Ser. No.07/678,916, filed Apr. 1, 1991, now abandoned.

This invention relates to recombinant DNA technology. In a particularaspect, the present invention relates to novel yeast strains producedemploying recombinant techniques, and novel DNA sequences encodingproteins involved in proteolytic processing, as well as novelauxotrophic marker proteins. In another aspect, the present inventionrelates to methods of producing recombinant products, especiallyrecombinant products which are susceptible to proteolytic degradation.

BACKGROUND OF THE INVENTION

Strains of the genus Pichia have been developed as an efficientexpression system for the production of recombinant products.Unfortunately, however, some protein products which are desirablyproduced by recombinant means (e.g., IGF-1, EGF, GRF, and the like) aresusceptible to degradation by proteases produced by the host organism.In such cases, even if high levels of the desired product are expressed,reduced product recoveries are sometimes realized due to degradation ofthe product in the presence of certain of the host strain's proteolyticenzymes. Product recovery is further complicated by the presence ofvarious proteolysis degradation products.

It would be desirable, in view of the excellent performance of thePichia-based expression system for the production of many recombinantproducts, to reduce or eliminate certain proteolytic activities ofPichia. This would reduce the likelihood of degradation ofprotease-sensitive products when produced in recombinant Pichia hosts.Reduced likelihood of degradation would result in an enhanced ability toexpress and recover such products in substantially intact form.

Various techniques can be applied in an effort to reduce or eliminatethe problem of proteolytic degradation of recombinantly producedproducts. For example, one could modify the conditions under whichrecombinant strains are grown so as to inhibit protease activity. Thiscould be accomplished, for example, by adjusting the pH of the mediumsufficiently to inhibit the action of various proteases. This approach,however, may affect the ability of the host organism to express certainrecombinant products (as well as the stability of the resulting product,once expressed). Moreover, this approach is limited only to its effecton extracellular proteolysis.

Alternatively, one could attempt to modify or eliminate some or all ofthe host organism's processing enzymes which are responsible for theproteolytic activity which degrades recombinantly produced,proteolytically sensitive products. Proteolytic processes in eukaryoticorganisms are, however, quite complicated and involved. Thus, it is notpossible to predict if elimination and/or modification of one or more ofthe enzyme(s) that are involved in proteolytic processing pathways willhave an impact on the viability of the host cells, and/or the stabilityof the recombinantly produced products.

Some of the proteolytic activities of the yeast S. cerevisiae have beencharacterized. Proteinase A, for example, is encoded by the S.cerevisiae PEP4 gene. Proteinase A is a vacuolar, aspartyl proteasecapable of self-activation, as well as subsequent activation ofadditional vacuolar proteases, such as carboxypeptidase Y, andproteinase B. Although carboxypeptidase Y appears to be completelyinactive prior to proteinase A-mediated proteolytic processing of theenzyme, proteinase B (encoded by the PRB-1 gene of S. cerevisiae)reportedly is approximately 50% bioactive in its precursor form (i.e.,the form that exists prior to proteinase A-mediated processing of theenzyme).

S. cerevisiae and filamentous fungi deficient in proteolytic activityhave previously been described. Such strains have been used for therecombinant expression of heterologous peptides. These organisms,however, differ substantially from the methylotrophic yeast, Pichia. Forexample, unlike Saccharomyces or Aspergillus, Pichia cells used for therecombinant expression of heterologous peptides are typically grown athigh cell density. High cell density growth is made possible, at leastin part, by selection of strains which minimize the occurrence offoaming during the fermentation process (which is accomplished byselecting for cells which produce large amounts of endo- andexo-proteases, which reduce foaming by reducing the size of proteinssecreted into the media). Furthermore, while growth at high cell densityenables the production of heterologous peptides in remarkably highyields, growth at high cell density also provides for a relatively highlevel of vacuolar proteases in the fermentation media (since ˜1% ofcells typically undergo lysis during yeast fermentation, the high celldensity process is accompanied by the release of substantial quantitiesof cellular material into the media, including vacuolar proteases).Therefore, during the production of heterologous peptides in a high celldensity process, some of the secreted, heterologous peptides produced byPichia could be subjected to substantial proteolysis.

Furthermore, since there are numerous metabolic and physiologicaldifferences between Saccharomyces, Aspergillus, and Pichia, it cannot beexpected that the proteolytic processing systems of these variousorganisms are necessarily similar. Indeed, very little is presentlyknown regarding the types of proteolytic activities present in Pichia.

SUMMARY OF THE INVENTION

In accordance with the present invention, we have isolated andcharacterized genes involved in proteolytic processes of species of thegenus Pichia. The availability of such genes has enabled the generationof strains of Pichia which are deficient in proteolytic activity, whichstrains are useful as hosts for the expression of proteolyticallysensitive products.

We have found that strains of Pichia which have been modified so as tobe defective in proteolytic activity, relative to wild-type Pichiacells, are excellent hosts for the expression of recombinant constructsencoding proteolytically sensitive products. The advantage of highlevels of recombinant product expression possible with the powerfulPichia expression system, coupled with the low level of proteolyticactivity in the invention host cells provides a highly efficientexpression system for the production of proteolytically sensitiveproducts.

In accordance with another embodiment of the present invention, we haveisolated and characterized the gene which encodes the Pichiaorotidine-5'-phosphate decarboxylase protein (i.e., the URA3 genes). Theavailability of this gene, in combination with strains of Pichia whichare Ura⁻, provides a particularly useful selection system for use inproducing recombinant strains of Pichia which are deficient inproteolytic activity. Such Ura⁻ strains are also useful as hosts fortransformation with recombinant DNA constructs, which are then used forthe recombinant expression of a variety of heterologous products.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a restriction map of plasmid pEP202.

FIG. 2 is a restriction map of plasmid pEP205.

FIG. 3 is a restriction map of plasmid pEP301.

FIG. 4 is a restriction map of plasmid pDR401.

FIG. 5 is a restriction map of plasmid pPU201.

FIG. 6 is a restriction map of plasmid pPU202.

FIG. 7 is a restriction map of plasmid pPU203.

FIG. 8 is a restriction map of plasmid pPU205.

FIG. 9 is a restriction map of plasmid pPU206.

FIG. 10 is a restriction map of plasmid pDR421.

FIG. 11 summarizes the steps employed in the construction of pDR601 andpDR602.

FIG. 12 is a restriction map of plasmid pDR601.

FIG. 13 is a restriction map of plasmid pDR602.

FIG. 14 is a restriction map of plasmid pDL521.

FIG. 15 is a restriction map of plasmid pDR911.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided an isolatedDNA fragment obtained from a strain of the genus Pichia which comprisesa gene encoding a protein which, directly or indirectly, influences theproteolytic activity of said strain.

In accordance with another embodiment of the present invention, there isprovided a method of producing modified strain(s) of the genus Pichiawhich are deficient in proteolytic activity, relative to host strain(s)of the same species which are not so modified, said method comprising:

contacting said host strain(s) with a modified form of theabove-described gene, wherein said modification renders the geneincapable of producing functional product, or alters the ability of thegene product to influence proteolytic activity, wherein said contactingis carried out under conditions suitable for the site-directedintegration of said modified form of the above-described gene into thegenome of said host strain(s), wherein said site-directed integrationoccurs at the specific locus of said gene which encodes said proteinwhich influences proteolytic activity.

In accordance with yet another embodiment of the present invention,there are provided strains of the genus Pichia which are deficient inproteolytic activity. Such strains can be produced in a variety of ways,with the above-described method being the presently preferred way ofproducing such strains.

In accordance with still another embodiment of the present invention,there is provided a method for the expression of proteolyticallysensitive recombinant product(s), said method comprising expressing saidproteolytically sensitive product(s) in the above-described Pichia cellswhich are deficient in proteolytic activity.

In accordance with a further embodiment of the present invention, thereis provided an isolated DNA fragment obtained from a species of thegenus Pichia which comprises the orotidine-5'-phosphate decarboxylasegene.

In accordance with a still further embodiment of the present invention,there is provided a yeast cell of the genus Pichia as a host capable ofbeing transformed with recombinant DNA material, wherein said host isdefective in the orotidine-5'-phosphate decarboxylase gene.

As employed herein, the term "proteolytic activity" refers to any one ormore of the enzyme activities displayed by enzymes involved in theproteolytic pathway. Proteolytic activities include proteinase Aactivity, proteinase B activity, carboxypeptidase Y activity,carboxypeptidase S activity, aminopeptidase C activity, dipeptidylaminopeptidase activity, proteinase D activity, proteinase E activity,and the like.

In accordance with one embodiment of the present invention, the Pichiagene that encodes a protein which, directly or indirectly, influences atleast the carboxypeptidase Y activity of strains of the genus Pichia hasbeen identified and isolated from a species of the genus Pichia. Thisgene is referred to herein, for convenience, as the Pichia PEP4 gene,based on the existence of some similarity between this gene and the S.cerevisiae PEP4 gene. It should be recognized, however, that thenucleotide sequences of the Pichia gene and the Saccharomyces genediffer substantially, as would be expected since the two species aresubstantially different. The novel Pichia PEP4 gene has the amino acidsequence encoded by Sequence ID No. 1. A fragment containing sequencesencoding this novel gene can be readily obtained for easy handling froma variety of sources. One such source is the approximately 10.6 kbpEcoRI fragment of plasmid pEP202 (see FIG. 1), or alternatively, theapproximately 2.7 kbp EcoRI-SacI fragment of plasmid pEP301 (see FIG.3). The proteinase A gene of the present invention can be furthercharacterized by reference to the amino acid sequence set forth inSequence ID No. 2. Any nucleic acid sequence which encodes substantiallythe same amino acid sequence as set forth in Sequence ID No. 2 can beemployed in the practice of the present invention. An exemplary nucleicacid sequence which encodes the above-described amino acid sequence isset forth in Sequence ID No. 1.

The Pichia gene that encodes a protein which, directly or indirectly,influences the proteolytic activity of strains of the genus Pichia canbe modified in a variety of ways, so as to render the gene incapable ofproducing functional product, or so as to alter the ability of the geneproduct to influence the proteolytic activity of said Pichia strain(s).Those of skill in the art recognize that there are many methods for themodification of the above-described gene. For example, the codingsequence can be mutated to modify the amino acid sequence of the proteinencoded by the gene. Alternatively, various portions of the codingsequence can be deleted from the gene. The deletion need only besufficient to render the expressed product (if it is still capable ofbeing expressed) non-functional. Thus, a deletion of even onenucleotide, by throwing the remaining coding sequence out of readingframe, can render a product, if still capable of expression,non-functional. Of course, larger deletions can result in a completelack of expression of product, or can cause a substantially modifiedproduct to be expressed, and such a product is likely to have verydifferent proteolytic properties, if any, relative to product producedby intact gene. As yet another alternative, additional sequences can beinserted into the coding sequence to disrupt the reading frame of thegene of interest, which would cause a dramatically altered product to beexpressed, or a complete lack of expression of the product.

A particularly convenient method for the modification of the Pichia genethat encodes a protein which, directly or indirectly, influences theproteolytic activity of strains of the genus Pichia is to insert anauxotrophic marker gene into said Pichia gene, thereby disrupting thePichia gene. Such auxotrophic marker genes can be selected from thePichia or Saccharomyces HIS4 gene, the Pichia or Saccharomyces ARG4genes, the Pichia or Saccharomyces URA3 genes, and the like.

Strains of Pichia deficient in proteolytic activity can be prepared in avariety of ways. The presently preferred method involves modifying, in asuitable host, genes of the present invention (which genes, in theirunmodified form, encode a product which, directly or indirectly, affectthe proteolytic activity of strains of the genus Pichia). Alternatively,host strains can be subjected to random (i.e., non-selective)mutagenesis, then screened to select for mutants which are deficient inproteolytic activity. This is not presently preferred because randommutagenesis is a non-selective process, which requires extensivescreening and selection in order to identify a well-characterizedmutant. In addition, there is the possibility of producing strains whichcontain multiple defects, as opposed to strains containing a single,well defined defect.

When proteolytically deficient strains are produced by modifying thegene of the invention in a host, such modifying is carried out, forexample, by introducing a modified gene under transformation conditionssuitable for the site-directed integration of the modified gene into thegenome of the host at the specific locus of such gene which encodes aprotein which influences proteolytic activity (i.e., the target gene).Integration will replace or alter the host's endogenous gene. Aconvenient means to introduce the modified gene into the target locus ofa yeast host is to include the modified gene in a linear DNA fragmenthaving ends homologous to two separate portions of the intact genewithin the host. This will direct, upon transformation, that homologousrecombination occur at the specific locus of the gene whose expressionproduct influences proteolytic activity.

When Pichia strains deficient in proteolytic activity are prepared bythe preferred method described above (i.e., by introducing a modifiedgene of the invention into a suitable host by site-directed integrationat the specific locus of the gene whose expression product influencesproteolytic activity, thereby replacing all or a portion of theendogenous gene with all or a portion of the modified gene), theendogenous gene is said to be disrupted. As used herein, the term gene"disruption" refers to any manipulation of the target locus thatultimately results in the presence of a gene that does not yield afunctional product, or that yields a product with altered function.Disruption can, therefore, result from the presence of added sequence(e.g., by the introduction of auxotrophic marker, or by the introductionof any sequence which causes a shift in the reading frame), the loss ofnucleotides from the target gene (e.g., by deletion), or other mutationsof the target gene. For the preferred method of preparing Pichia strainsdeficient in proteolytic activity, gene disruption is achieved by geneaddition, gene replacement, or a combination of addition and replacementreferred to herein as "pop-in-pop-out". In gene replacement, theendogenous target gene is physically removed from the target locus, andreplaced with the modified gene. This is accomplished by transformingthe host with a linear fragment having ends which are homologous to the5' and 3' ends of the target gene, respectively. Gene addition involvesadding the transforming DNA to the endogenous target gene. Depending onthe manner in which the modified gene of the transforming DNA wasaltered, gene addition can result in the presence of either twonon-functional copies of the target gene, or one functional and onenon-functional copy of the target gene. Each of the two copies consistsof a portion of the endogenous gene, and a portion of the transformingDNA. If a functional copy of the target gene remains after geneaddition, it can then be removed by homologous recombination between thetwo copies of the target gene. The combination process of gene additionfollowed by homologous recombination constitutes the pop-in-pop-outprocess.

Methods of transforming yeast of the genus Pichia, as well as methodsapplicable for culturing such yeast cells, are known generally in theart.

According to the invention, constructs containing the above-describedmodified gene are transformed into Pichia cells either by thespheroplast technique, described by Cregg et al., in Mol. Cell. Biol.5:3376 (1985) and U.S. Pat. No. 4,879,231, or by the whole-cell lithiumchloride yeast transformation system Ito et al., Agric. Biol. Chem.48:341 (1984)!, with modification necessary for adaptation to Pichia SeeEuropean Patent Application No. 312,934; also available as U.S. Pat. No.4,929,535!. The whole-cell lithium chloride method is frequently moreconvenient in that it does not require the generation and maintenance ofspheroplasts. However, for the purpose of the present invention, thespheroplast method is preferred because the spheroplast method isgenerally a more efficient means of transformation.

Those of skill in the art recognize that host Pichia strains fortransformation with the above-described modified gene can be wild-typePichia cells, which upon transformation with a defective gene from theproteolytic pathway, could be screened for reduced proteolytic activity.The host strains employed can have one or more defects therein, toassist in the identification and selection of desired transformants.

Preferred hosts employed for transformation with a modified form of thegene which encodes a protein which, directly or indirectly, influencesthe proteolytic activity of strains of Pichia, is a strain which isdefective in at least one auxotrophic marker gene. The use of such hostorganisms is preferred because simultaneous transformation of such ahost with the modified form of the invention gene and an auxotrophicmarker gene enables rapid selection of strains which have incorporatedthe transforming DNA, and thus, should have a disrupted form of the genewhich encodes a protein which directly or indirectly influences theproteolytic activity of the host.

Exemplary auxotrophic marker genes useful in the practice of the presentinvention (i.e., marker genes that are defective in the preferred hoststrains employed herein) include the histidinol dehydrogenase gene, theargininosuccinate lyase gene, or the orotidine-5'-phosphatedecarboxylase gene, and the like. When employing such host strains inthe transformation of Pichia, the above-described modified gene,contained in a linear DNA fragment, is preferably associated with anintact form of the auxotrophic marker gene for which the host strain isdefective, e.g., the auxotrophic marker gene either is contained withinthe modified gene, or is located 5' or 3' of the modified gene on thetransforming linear DNA fragment. Exemplary host strains contemplatedfor use in the practice of the present invention include thehis4-defective Pichia strain, GS115 (ATCC 20864), the arg4-defectivePichia strain, GS190, the his4/ura3-defective Pichia strain, GS4-2, thehis4/arg4-defective Pichia strain PPF1 (NRRL Y-18017; see U.S. Pat. No.4,812,405), and the like. An exemplary fragment of DNA which containsthe above-described modified gene having inserted therein a functionalgene encoding histidinol dehydrogenase can be obtained from theapproximately 5.3 kbp SacI-EcoRI fragment of plasmid pDR401. Anotherexemplary fragment of DNA which contains a modified form of theabove-described gene (located 5' of a functional gene encodingorotidine-5'-phosphate decarboxylase) can be obtained from theapproximately 5.0 kbp BglII fragment of plasmid pDR421.

A particularly advantageous application of the Pichia strains of thepresent invention (i.e., strains which are deficient in proteolyticactivity) is the expression of proteolytically sensitive recombinantproducts, such as, for example, epidermal growth factor (EGF), growthhormone releasing factor (GRF), insulin-like growth factor-1 (IGF-1),and the like. When expressed in recombinant Pichia strains which aredeficient in proteolytic activity, the resulting recombinant product issubjected to a reduced level of proteolytic activity, due tomodifications in the proteolysis apparatus of the host organism. Suchproteolytically deficient Pichia expression systems for the productionof proteolytically sensitive products can be generated in a variety ofways. For example, Pichia host strains can be rendered proteolyticallydeficient, as described hereinabove, and then further transformed withDNA encoding a heterologous protein of interest (especially aproteolytically sensitive protein). Alternatively, a recombinant Pichiastrain already bearing DNA encoding a heterologous protein of interestcan thereafter be rendered proteolytically defecient, for example, asdescribed hereinabove. As yet another alternative, a Pichia strain couldbe co-transformed with the above described modified gene and a DNAencoding a heterologous, proteolytically sensitive protein of interest.

The use of strains of the genus Pichia as host strains in therecombinant expression of peptide products has previously been describedin great detail.

The presently preferred yeast species for use in the practice of thepresent invention is Pichia pastoris, a known industrial yeast strainthat is capable of efficiently utilizing methanol as the sole carbon andenergy source.

There are a number of methanol-responsive genes in methylotrophic yeast,the expression of each being controlled by methanol-responsiveregulatory regions (also referred to as promoters). Any of suchmethanol-responsive promoters are suitable for use in the practice ofthe present invention. Examples of specific regulatory regions includethe promoter for the primary alcohol oxidase gene from Pichia pastorisAOX1, the promoter for the secondary alcohol oxidase gene from P.pastoris AOX2 (P. pastoris is known to contain two functional alcoholoxidase genes: alcohol oxidase I (AOX1) and alcohol oxidase II (AOX2);the coding portions of the two AOX genes are closely homologous at boththe DNA and the predicted amino acid sequence levels and share commonrestriction sites; the proteins expressed from the two genes havesimilar enzymatic properties but the promoter of the AOX1 gene is moreefficient and gene products are frequently more highly expressedtherefrom), the promoter for the dihydroxyacetone synthase gene from P.pastoris (DAS), the promoter for the P40 gene from P. pastoris, thepromoter for the catalase gene from P. pastoris, the promoter for theformaldehyde dehydrogenase gene from P. pastoris, the promoter for theformate dehydrogenase gene from P. pastoris, and the like.

The presently preferred promoter region employed to drive expression ofa gene encoding a proteolytically sensitive product, in P. pastorishosts, is the promoter of the methanol-regulated primary alcohol oxidasegene of P. pastoris. The AOX1 gene, including its promoter, has beenisolated and thoroughly characterized; see Ellis et al., Mol. Cell.Biol. 5: 1111 (1985) and U.S. Pat. No. 4,855,231.

The presently preferred expression cassette used in transforming Pichiacells for the generation of recombinant protein-expressing strainscomprises, in the reading frame direction of transcription, thefollowing DNA sequences:

(i) a promoter region of a methanol-responsive gene of a methylotrophicyeast,

(ii) a DNA sequence encoding a polypeptide consisting of:

(a) an optional secretion signal sequence, and

(b) a heterologous protein of interest; and

(iii) a transcription terminator functional in a methylotrophic yeast;

wherein said DNA sequences are operationally associated with one anotherfor transcription of the sequences encoding said polypeptide. DNAsequences encoding a secretion signal sequence which are optionallycontained in expression vectors used in the practice of the presentinvention include the DNA encoding the native secretion signal sequenceassociated with the proteolytically sensitive product, the DNA encodingthe S. cerevisiae α-mating factor (αMF) leader sequence, (including aDNA sequence encoding the processing site, lys-arg), and the like.

The transcription terminator functional in a methylotrophic yeast usedin accordance with the present invention has either (a) a subsegmentwhich provides a polyadenylation signal and polyadenylation site in thetranscript, and/or (b) a subsegment which provides a transcriptiontermination signal for transcription from the promoter used in theexpression cassette. The term "expression cassette" as used herein, andthroughout the specification and claims, refers to a DNA sequence whichincludes sequences functional for the expression process. The entiretranscription terminator is taken from a protein-encoding gene, whichmay be the same or different from the gene which is the source of thepromoter.

In the DNA constructs of the present invention, used to transform hostsfor recombinant expression of proteolytically sensitive products, thesegments of the expression cassette(s) are said to be "operationallyassociated" with one another. The DNA sequence encoding proteolyticallysensitive products is positioned and oriented functionally with respectto the promoter, the secretion signal sequence, if employed, and thetranscription terminator. Thus, the polypeptide-encoding segment istranscribed, under regulation of the promoter region, into a transcriptcapable of providing, upon translation, the desired polypeptide.Appropriate reading frame positioning and orientation of the varioussegments of the expression cassette are within the knowledge of personsof ordinary skill in the art; further details are given in the Examples.

For the practice of the present invention it is preferred that hosts forthe recombinant expression of proteolytically sensitive products betransformed with multiple copies of the above-described expressioncassettes contained on one DNA fragment, preferably in a head-to-tailorientation.

In addition, when DNA constructs according to the invention are used totransform hosts for the recombinant expression of proteolyticallysensitive products by site-directed integration, the expressioncassette-containing construct is a linear DNA fragment that is directedto the desired locus of the host to effect integration of the DNAfragment therein. One-step gene integrations are usually successful ifthe DNA to be introduced has as little as 0.2 kb homology with thefragment locus of the target gene; it is however, preferable to maximizethe degree of homology for efficiency.

The DNA constructs used according to the invention to transform hostsfor the recombinant expression of proteolytically sensitive productsoptionally further comprise a selectable marker gene, in addition to oneor more expression cassettes. For this purpose, any selectable markergene functional in methylotrophic yeast may be employed, i.e., any genewhich confers a phenotype upon methylotrophic yeast cells, therebyallowing them to be identified and selectively grown from among a vastmajority of untransformed cells. Suitable selectable marker genesinclude, for example, selectable marker systems composed of anauxotrophic mutant P. pastoris host strain and a wild-type biosyntheticgene which complements the host's defect. For transformation of His4⁻ P.pastoris strains, for example, the S. cerevisiae or P. pastoris HIS4gene may be employed, or for transformation of Arg4⁻ mutant P. pastorisstrains, the S. cerevisiae ARG4 gene or the P. pastoris ARG4 gene may beemployed, or for transformation of Ura3⁻ mutant P. pastoris strains, theS. cerevisiae URA3 gene or the P. pastoris URA3 gene may be employed.

In addition, DNA constructs used to transform hosts for the recombinantexpression of proteolytically sensitive products according to thisaspect of the invention optionally further comprise selectable markergenes which are functional in bacteria. Thus, any gene can be used whichconfers a phenotype on bacteria that allows transformed bacterial cellsto be identified and selectively grown from among a vast majority ofuntransformed cells. This additional selectable marker enables DNA ofthe invention to be transformed into bacteria such as E. coli foramplification. Suitable selectable marker genes include the ampicillinresistance gene (Amp^(r)), tetracycline resistance gene (Tc^(r)), andthe like.

When it is contemplated to pass DNA of the invention though bacterialcells, it is desirable to include in the DNA construct a bacterialorigin of replication, to ensure the maintenance of the invention DNAfrom generation to generation of the bacteria. Exemplary bacterialorigins of replication include the fl-ori, colisin, col El, and thelike.

The term "expression vector", as employed herein, is intended to includevectors capable of expressing DNA sequences contained therein, wheresuch sequences are in operational association with other sequencescapable of effecting their expression, i.e., promoter sequences. Ingeneral, expression vectors usually used in recombinant DNA technologyare often in the form of "plasmids", i.e., circular, double-stranded DNAloops, which in their vector form are not bound to the chromosome. Inthe present specification the terms "vector" and "plasmid" are usedinterchangeably. However, the invention is intended to include otherforms of expression vectors as well, which function equivalently.

Methods of transforming yeast of the genus Pichia, as well as methodsapplicable for culturing such yeast cells, are known generally in theart.

According to the invention, constructs containing the above-describedmodified gene and/or expression cassettes encoding the production ofheterologous, proteolytically sensitive products are transformed intoPichia cells either by the spheroplast technique, or by the whole-celllithium chloride yeast transformation system, as described above.

Transformed strains, which are of the desired phenotype and genotype,are grown in fermentors in either batch or continuous mode. For thelarge-scale production of recombinant DNA-based products inmethylotrophic yeast, a three-stage, high cell-density fermentationsystem is the presently preferred fermentation protocol employed. In thefirst, or growth stage, expression hosts are cultured in defined minimalmedium with an excess of a non-inducing carbon source (e.g., glycerol).When grown on such carbon sources, heterologous gene expression iscompletely repressed, which allows the generation of cell mass in theabsence of heterologous protein expression. It is presently preferred,during this growth stage, that the pH of the medium be maintained atabout 5, because the P. pastoris cells generally prefer a pH of about 5for optimal growth. Next, a short period of non-inducing carbon sourcelimitation growth is allowed to further increase cell mass and derepressthe methanol-responsive promoter. The pH of the medium during thislimitation growth period is maintained at an appropriate pH value (theactual pH employed is a function of the particular host strain used forexpression and the specific product being expressed).

Subsequent to the period of growth under limiting conditions, methanolis added in the fermentor either on a continuous basis, with concurrentremoval of product via the broth; or on a batch-wise basis whereinmethanol is added at such a rate that the methanol content of the brothis maintained at a low level (referred to herein as "methanol excessfed-batch mode"). The addition of methanol induces the expression of thegene driven by a methanol-responsive promoter. This third stage isreferred to as the production stage, because it is at this stage thatthe majority of the recombinant product is expressed. The pH of themedium during the production stage is maintained at an appropriate pHvalue (the actual pH employed is a function of the particular hoststrain used for expression and the specific product being expressed).

The term "culture" means a propagation of cells in a medium conducive totheir growth, and all sub-cultures thereof. The term "subculture" refersto a culture of cells grown from cells of another culture (sourceculture), or any subculture of the source culture, regardless of thenumber of subculturings which have been performed between the subcultureof interest and the source culture.

According to a preferred embodiment of the present invention, theheterologous protein expression system used for the production ofproteolytically sensitive products utilizes the promoter derived fromthe methanol-regulated AOX1 gene of P. pastoris, which is very efficientand tightly regulated. This gene can be the source of the transcriptionterminator as well. The presently preferred expression cassettecomprises, operationally associated with one another, the P. pastorisAOX1 promoter, optional DNA encoding a secretion signal sequence, a DNAsequence encoding a proteolytically sensitive product (e.g., matureIGF-1, EGF, GRF, and the like), and a transcription terminator derivedfrom the P. pastoris AOX1 gene. Preferably, two or more of suchexpression cassettes are contained on one DNA fragment, in head-to-tailorientation, to yield multiple expression cassettes on a singlecontiguous DNA fragment.

The presently preferred host cells to be transformed with multipleexpression cassettes are P. pastoris cells having at least one mutationthat can be complemented with a marker gene present on a transformingDNA fragment. Preferably His4⁻ (GS115) or Arg4⁻ (GS190) singleauxotrophic mutant P. pastoris strains are employed, or His4⁻ /Ura3⁻(GS4-2) or His4⁻ /Arg4⁻ (PPF1) double auxotrophic mutant P. pastorisstrains are employed.

The fragment containing one or more expression cassette(s) is insertedinto a plasmid containing a marker gene complementing a metabolic defectin the host, and optionally containing additional sequences such asbacterial marker genes, yeast DNA sequences which direct vectorintegration, and the like.

In accordance with a specific embodiment of the present invention, thereis provided an isolated DNA fragment obtained from a species of thegenus Pichia which comprises the orotidine-5'-phosphate decarboxylasegene. The orotidine-5'-phosphate decarboxylase gene is frequentlyreferred to as URA3. It can be used, for example, to complementURA3-deficient strains. Another use for the novel gene is the ability totarget DNA into a specific locus of the Pichia genome (i.e., into theURA3 locus). This novel gene has the amino acid sequence set forth inSequence ID No. 4. Alternatively, this novel gene can be characterizedas encoding a protein having substantially the same amino acid sequenceas set forth in Sequence ID No. 4. While those of skill in the artrecognize that the above-referenced amino acid sequence can be encodedby a variety of nucleotide sequences, a presently preferred nucleotidesequence encoding the above-referenced amino acid sequence issubstantially the same as that set forth in Sequence ID No. 3.

In accordance with another specific embodiment of the present invention,there are provided yeast cells of the genus Pichia as a host capable ofbeing transformed with recombinant DNA material, wherein the host isdefective in the orotidine-5'-phosphate decarboxylase gene. Host strainsdefective in the URA3 gene can be used for transformation with DNAcontaining an intact form of the URA3 gene, thereby enabling a readydetermination of whether the desired transformation event has occurred(by return of successfully transformed cells to uracil prototrophy).

The combination of Ura3⁻ Pichia strains and the Pichiaorotidine-5'-phosphate decarboxylase marker gene provides a particularlyuseful selection system for use in producing recombinant strains ofPichia deficient in proteolytic activity. Such a selection system isreferred to herein as a "bidirectional selection process". Applicationof this selection system for the generation of Pichia strains which aredeficient in proteolytic activity is carried out as follows:

A Ura3⁻ host is transformed with a DNA construct containing a modifiedform of a gene encoding a protein involved in the Pichia proteolyticpathway, and the URA3 gene. Site-directed integration of thetransforming DNA by gene addition (i.e., "pop-in") yields one functionaland one non-functional gene at the locus of the gene which directly orindirectly influences proteolytic activity, as well as an intact URA3gene. Strains which incorporate the URA3 gene are identified by positiveselection (using techniques well known to those of skill in the art,e.g., by growing the strains on minimal media lacking uracil andselecting those strains capable of growth on such media). Theconfiguration of the functional, non-functional and URA3 genes at thelocus of the gene which encodes a protein which influences proteolyticactivity enables recombination to occur between the functional andnon-functional genes, resulting in the loss of one of these genes andthe URA3 gene (i.e., "pop-out"). Thereafter, it is possible topositively select for strains lacking a functional URA3 gene by platingcells on medium containing a non-toxic analog of a uracil pathwayintermediate, 5-fluoro-orotic acid (5-FOA), which, when metabolized byUra3⁻ strains, produces a compound toxic to the cells. Because Ura3⁻strains blocked at a specific point in the uracil pathway do notmetabolize 5-FOA, they are not subjected to its toxic effects, and canthus be referred to as "5-FOA resistant". In contrast, Ura3⁺ strainsmetabolize 5-FOA to produce a toxic compound which will prevent growthof the Ura3⁺ cells. The resulting Ura3⁻ cells that also lack thefunctional target gene are deficient in proteolytic activity. Becausethe Ura3⁻ phenotype is restored, the resulting cells can be transformedagain using the URA3 gene as a selectable marker.

The ability to positively select strains lacking a functional URA3 geneemploying a toxic analog of a uracil pathway intermediate allows the useof this very convenient "pop-out" method for imparting multiplephenotypic changes in Pichia hosts.

Ura3⁻ strains which are also deficient in proteolytic activity, relativeto the proteolytic activity present in wild-type strains of the samespecies, are particularly useful for transformation with expressionvectors which contain an intact form of the URA3 gene, and a geneencoding a proteolytically sensitive product (either as part of the samevector, or as a second vector which is transformed into the host). Thosetransformants which return to uracil prototrophy (which can be readilydetermined by simple screening procedures) should have incorporatedtherein the gene encoding a proteolytically sensitive product, and thuswould be directly applicable to product expression.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES EXAMPLE I ISOLATION OF THE P. PASTORIS PEP4 GENE

The P. pastoris PEP4 gene was identified in a bacteriophage lambda-basedEMBL3 P. pastoris genomic DNA library by its ability to hybridize with aradiolabeled fragment of the homologous Saccharomyces cerevisiae PEP4gene. The P. pastoris PEP4 gene was cloned by isolating positive plaquescontaining the hybridizing recombinant phage DNA.

A. Construction of a P. pastoris EMBL3 Genomic DNA Library

Bacteriophage A was used as a vehicle for cloning the P. pastoris gene.Fragments of a partial Sau3A digest of P. pastoris genomic DNA wereinserted into the bacteriophage λ vector EMBL3, which contains elementsof the bacteriophage λ genome essential for propagation of therecombinant DNA in bacterial hosts. The P. pastoris DNA-containing EMBL3vectors were packaged in vitro into infectious virions to yield abacteriophage λ P. pastoris genomic DNA library. Amplification of thelibrary was achieved by propagation of the recombinant DNA inEscherichia coli host cells that had been infected with the recombinantvirus.

EMBL3 Frischauf, A.-M. et al. (1983). J. Mol. Biol. 170:827! is areplacement vector capable of incorporating fragments of genomic DNAranging in size from 9 to 23 kb. This vector contains a segment ofnonessential bacteriophage λ DNA (stuffer fragment) that is delineatedby a pair of restriction sites (Bam HI/EcoRI) located at both ends ofthe segment in opposite orientations (i.e., 5'BamHI-EcoRI-stuffer-EcoRI-BamHI 3'). Foreign DNA fragments containingBamHI-compatible ends (e.g., Sau3A termini) are incorporated into thevector by replacement of the stuffer fragment.

Pichia pastoris genomic DNA (from strain NRRL Y-11430, from the NorthernRegional Research Center, Peoria, Ill.) isolated using a glass rod swirltechnique Cregg et al. Mol. Cell. Biol. 5:3376-3385 (1985)! was digestedwith Sau3A at an effective concentration of 0.1 u/μg in 7, 14, 21 and 28minute incubations conducted at 37° C. An aliquot from each incubationmixture was electrophoretically separated on a 1% agarose gel todetermine the sizes of the digested DNA fragments. Digests incubated for7 and 14 minutes appeared to consist primarily of 9-23 kb fragments.These digests were pooled and ligated to EMBL3 vector arms, prepared asdescribed below.

EMBL3 vector arms were prepared by double digestion of the vector(obtained from EMBL3 Cloning Kit, Stratagene Cloning Systems, San Diego,Calif.; catalog #241211) with BamHI and EcoRI. The small BamHI/EcoRIlinker that separates the arms from the stuffer fragment was removedfrom the digest by selective precipitation with ethanol. Because thearms end in BamHI termini and the stuffer sequence is contained in anEcoRI fragment, the arms were unable to religate to the stufferfragment. Therefore, following removal of the BamHI/EcoRI linker, it wasnot necessary to separate the arms from the stuffer fragment prior toligation of the arms and the genomic DNA inserts. Ligation of theSau3A-digested Pichia genomic DNA (0.5 μg) to 1 μg of EMBL3 pre-digestedarms was accomplished by incubation of the 5-μl reaction mixture at 4°C. for two days.

The recombinant bacteriophage λ DNA prepared by ligation of P. pastorisgenomic DNA fragments and EMBL3 vector arms was packaged in vitro usingcommercial packaging extracts (Stratagene EMBL3 Cloning Kit). Theefficiency of packaging was determined by plating an aliquot of thepackaged library and the E. coli lysogenic host strain VSC 257 onto NZY(5 g NaCl, 2 g MgSO₄.H₂ O, 5 g yeast extract, 10 g NZ amine and 20 gagar per liter) plates. The efficiency of packaging was calculated anddetermined to be 1.2×10⁶ plaques/μg.

The EMBL3-based P. pastoris genomic library was amplified by plating therecombinant phage along with the E. coli lysogenic host strain P2 392(provided in Stratagene EMBL3 Cloning Kit) which contains prophage P2.Wild-type bacteriophage do not grow in E. coli strain P2 392.Recombinant EMBL3-based bacteriophage, created by replacing the stufferfragment of EMBL3 with foreign DNA, lack two of the wild-type genes thatconfer P2 sensitivity, which were contained in the stuffer fragment.Therefore, the recombinant bacteriophage are able to grow well in thisP2-containing E. coli strain. The use of E. coli P2 392 as the hoststrain in the amplification ensured that only recombinant phage would bereproduced in the bacterial host. All of the plates encompassing theEMBL3-based P. pastoris genomic DNA library were overlayed with SMbuffer (5.8 g NaCl, 2 g MgSO₄.H₂ O, 50 ml 1M Tris.HCl, pH 7.5, and 5 ml2% gelatin per liter). After five hours, the supernatants were collectedand pooled, and the titer and genome equivalents were calculatedaccording to the manufacturer's instructions. The library containedapproximately 10 genome equivalents, and its titer was 6×10¹¹plaque-forming units/ml (pfu/ml).

B. Screening of the EMBL3 P. pastoris Genomic DNA Library Using the S.cerevisiae PEP4 Gene as a Probe

In order to adequately screen the Pichia genome for the PEP4 gene,50,000 recombinant phage and the E. coli lysogenic host strain LE 392(provided in Stratagene EMBL3 Cloning Kit) were plated onto four large150-mm plates. After 6-7 hours of growth, the plates were chilled to 4°C. Each plate was marked and duplicate plaque lifts of each plate wereprepared by placing nitrocellulose onto each plate. The filters weredenatured, neutralized, baked and probed with the S. cerevisiae PEP4gene a gel-purified, ³² P-labeled 4.0 kb fragment of S. cervisiae DNAcontaining the S. cerevisiae PEP4 gene obtained from the laboratory ofThomas Stevens, University of Oregon, Eugene, Ore.; see Rothman et al.,Proc. Natl. Acad. Sci. U.S.A. 83: 3248-3252 (1986)!. Hybridization wasconducted at 37° C. in a solution containing 30% formamide, 6×SSC,5×Denhardt's solution, 20 mM Tris.HCl, pH 8.0, 1 mM EDTA, 0.1% SDS and100 μg/ml salmon sperm DNA. After hybridization, the filters were washedthree times at room temperature using 2×SSC and 0.1% SDS. Followingthese initial washes, the filters were then washed twice at 55° C. using2×SSC and 0.1% SDS.

Fifteen positive plaques containing DNA that hybridized to the fragmentof the S. cerevisiae PEP4 gene were identified in duplicate fromautoradiograms of the filters. The area around each of the 15 positiveplaques was isolated and placed in SM buffer. Six of the isolates wereplated at dilutions of 10⁻⁵ and 10⁻⁷ with E. coli strain LE 392 ontosmaller 100-mm plates. Single plaque lifts of each plate were probedwith the S. cerevisiae PEP4 gene fragment under the same hybridizationand wash conditions used in the first plaque screening. In this secondround of screening, 12 positive plaques were detected on theautoradiogram. Nine of these single plaques were isolated and placed inSM buffer. Each of these nine plaques was plated at dilutions of 10⁻⁵and 10⁻⁷ with E. coli strain LE 392 onto small 100-mm plates. Again,single plaque lifts of each plate were probed with the S. cerevisiaePEP4 gene fragment under the same hybridization and wash conditions usedin the first two screenings. Each plate contained approximately 10-20plaques distributed evenly across the plate. Autoradiograms of thefilters revealed that every plaque on each plate hybridized to the PEP4probe.

Five separate plaques from different plates were isolated and placed inSM buffer. DNA from large-scale cultures of three of these isolates,designated 4721, 5111 and 5131, respectively, was prepared using theinduction method of bacteriophage isolation Maniatis, T., Fritsch, E. F.and Sambrook, J. Molecular Cloning, A Laboratory Manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., U.S.A. (1982)! inorder to identify, characterize and subclone the PEP4 gene contained inthe recombinant phage.

C. Characterization of the Insert in Isolates of the EMBL3 P. pastorisGenomic DNA Library that Hybridized to the S. cerevisiae PEP4 Gene

Recombinant phage DNAs of the three isolates referred to above of theEMBL3 Pichia genomic DNA library (4721, 5111 and 5131) were digestedwith various restriction endonucleases, separated on a 0.8% agarose geland visualized by ethidium bromide staining. In addition, 1 μl aliquotsof these digests were separated on a second agarose gel which wasblotted onto nitrocellulose and probed with the radiolabeled S.cerevisiae PEP4 gene fragment. Hybridization was conducted at 37° C. ina solution containing 30% formamide, 6×SSC, 5×Denhardt's solution, 20 mMTris.HCl, pH 8.0, 1 mM EDTA, 0.1% SDS and 100 μg/ml salmon sperm DNA.The filter was then washed in three 5-minute washes at room temperaturewith 2×SSC and 0.1% SDS followed by two 5-minute washes at 55° C. with2×SSC and 0.1% SDS.

Identical digests of DNA from two of the clones, 5111 and 5131, yieldedthe same pattern of restriction enzyme fragments, as determined byethidium bromide staining, whereas the same digest of DNA from the thirdclone, 4721, yielded a different fragment pattern. Analysis of therestriction enzyme fragments of DNA from each clone by Southern blothybridization to the S. cerevisiae PEP4 gene fragment revealed that thetwo classes of clones both contained a series of hybridizing fragmentsof the same size indicating that the two classes of clones had a commonoverlapping DNA sequence that hybridized with the probe.

D. Subcloning and Characterization of the Cloned P. pastoris PEP4 Gene

As determined by Southern blot hybridization of EcoRI-digested P.pastoris genomic DNA using the homologous S. cerevisiae PEP4 gene as aprobe, the P. pastoris PEP4 gene is contained within a 10.6 kb EcoRIfragment of the P. pastoris genome. Southern blot hybridization ofEcoRI-digested DNA of clone 4721, as described in Example IC, revealedthat it contained a 10.6 kb fragment that hybridized to the S. cervisiaePEP4 gene. To facilitate manipulation of the cloned P. pastoris PEP4gene, P. pastoris genomic DNA contained on an EcoRI fragment of DNA fromisolate 4721 was subcloned into pUC19. Clone 4721 (25 μg) was digestedwith EcoRI (60 units) in a total volume of 300 μl. The digested DNA wasseparated on a 0.65% agarose gel, and the 10.6 kb EcoRI fragment wasisolated with DE81 paper. The purified fragment was washed from thepaper with 400 μl of 1M NaCl and extracted with phenol/chloroform. TheDNA was then precipitated with ethanol and resuspended in water to atotal volume of 10 μl. Approximately 50 ng of the 10.6 kb fragment wereligated with an equal amount of pUC19 which had been cut with EcoRI anddephosphorylated. The ligation mixture was used to transform E. colistrain MC1061. Ampicillin-resistant colonies were selected and screenedby analysis of restriction enzyme digests of colony DNA for the presenceof the diagnostic 10.6 kb EcoRI fragment. A large-scale plasmidpreparation was made from a colony containing the correct plasmid, whichwas named pEP202. Plasmid pEP202 contains the complete P. pastoris PEP4gene (see FIG. 1).

To facilitate sequence analysis of the cloned P. pastoris PEP4 gene, aportion of the P. pastoris PEP4 gene was subcloned into pUC19. PlasmidpEP202 was digested with BamHI and EcoRI. The reaction mixture wasseparated on a 0.7% agarose gel, and the 0.45 kb BamHI fragment of DNA(see FIG. 2) was isolated using DE81 paper. The purified fragment wasligated to pUC19 (˜20 ng) that had been linearized by digestion withBamHI and dephosphorylated. The ligation mixture was used to transformE. coli strain MC1061. Transformants were selected for ampicillinresistance and screened by analysis of restriction enzyme digests ofcolony DNA for the presence of a single bamHI fragment. A single colonyarising from this transformation was found to contain the appropriateDNA construct, and was named pEP205 (see FIG. 2).

Sequence analysis of plasmid pEP202 identified a DNA sequence with ˜70%homology to the PEP4 gene of S. cerevisiae. The amino acid sequenceencoded by this DNA sequence of pEP202 is 69% homologous to that encodedby the S. cerevisiae PEP4 gene.

EXAMPLE II DEVELOPMENT OF A PEP4-DEFICIENT (Pep4⁻) STRAIN OF P. PASTORIS

A. Construction of the P. pastoris PEP4 Gene Disruption Vector pDR401

Vector pDR401 was constructed for use in developing a PEP4-deficient(Pep4⁻) strain of P. pastoris. This vector contains a defective P.pastoris PEP4 gene, which, when used to transform PEP4 strains of P.pastoris, integrates into the host genome by replacement of thewild-type PEP4 gene.

pDR401 was constructed in a two-step procedure as follows. In the firststep, the base vector in the construction of pDR401, base vector pEP301,was constructed from pEP202. VeCtor pEP301 consists of pUC19 sequencesand the cloned P. pastoris PEP4 gene from pEP202. Plasmid pEP202 (15 μg)was digested with SacI. A 5.5 kb SacI fragment (the fragment extendingfrom the SacI linker clockwise to the SacI site at ˜5:00, and containingall of the pUC19 sequence and the entire PEP4 gene; see FIG. 1) wasisolated from a 0.7% agarose gel using DE81 paper. The fragment waseluted from the paper with 400 μl of 1M NaCl, extracted with 400 μl ofphenol/chloroform and precipitated with ethanol. This DNA was thenligated with itself in a volume of 100 μl containing 1 μl of ligase and1 μl (˜10 ng) of DNA. The ligation mixture was incubated at roomtemperature for 1 hr and then used to transform E. coli strain MC1061.Ampicillin-resistant colonies were selected and screened by analysis ofrestriction enzyme digests of colony DNA for the presence of a single5.5 kb BglII fragment. Plasmid DNA was prepared from a transformedcolony of MC1061 that contained the correct plasmid, which was namedpEP301 (FIG. 3).

In the second step of the construction of pDR401, the P. pastoris HIS4gene was inserted into the PEP4-containing plasmid pEP301 to yield thefinal vector. The P. pastoris HIS4 gene was isolated on a 2.6 kb BglIIfragment derived from pYJ8ΔCla Cregg, J. et al. Mol. Cell. Biol.5:3376-3385 (1985)!. Plasmid pYJ8ΔCla (15 μg) was digested with BglIIand the digested DNA was separated on a 0.7% agarose gel. The HIS4gene-containing 2.6 kb fragment was isolated with DE81 paper, elutedwith 400 μl of 1M NaCl, extracted with 400 μl of phenol/chloroform,precipitated with ethanol and resuspended in 10 μl of water. Prior toinserting this 2.6 kb BglII fragment into the unique BglII site ofpEP301, approximately 20 μg of pEP301 were digested with BglII,dephosphorylated and extracted with phenol/chloroform. The 2.6 kbHIS4-containing fragment was then inserted into pEP301 by ligation ofapproximately 50 ng of the fragment to approximately 50 ng of theBglII-digested pEP301 in a total volume of 10 μl containing 1 μl ofbuffer, 1 μl of ligase and water. Ligation was conducted at roomtemperature for 3 hrs and the ligation mix was used to transform MC1061cells. Plasmid DNA prepared from an ampicillin-resistant colony wasdigested with BglII, SalI, BglII/SalI, PvuI, NcoI and KpnI to confirmthe construction of pDR401. The restriction fragment pattern wasconsistent with that expected for the correct plasmid pDR401 (see FIG.4). Plasmid pDR401 is pUC19 with the P. pastoris HIS4 gene inserted atthe unique BglII site within the PEP4 structural gene, thus disruptingit.

B. Transformation of his4 P. pastoris Strain GS115 with a Fragment ofpDR401

In order to create a pep4 strain of P. pastoris, the his4 PEP4 P.pastoris strain GS115 (ATCC 20864) was transformed with 20 μg of the 5.3kb EcoRI/SacI fragment of pDR401 according to the spheroplast method(see U.S. Pat. No. 4,879,231). This fragment of pDR401 consists of theHIS4 gene-containing defective pep4 gene. Transformant strains resultingfrom this type of integration are prototrophic and can be distinguishedfrom untransformed cells on this basis. The frequency of transformationwas approximately 10³ μg⁻¹ DNA.

C. Characterization of Transformants

1. Analysis of transformant carboxypeptidase Y activities

His⁺ transformants were subsequently analyzed for carboxypeptidase Yactivity using a colony overlay colorimetric screening procedure seeJones, E. in Genetics 85: 23-33 (1977)!. In this assay, the His⁺transformant cells were released from the transformation agar plates andgrown on YEPD (yeast extract, 1% peptone, 2% dextrose and 2% agar)plates at a density of ˜300 colonies per plate. The plates wereoverlayed with 0.6% agarose containing 40% dimethylformamide (DMF) topermeabilize the cells, and 1.2 mg/ml of the substrate APNE (N-acetyl DLphenylalanine β-napthyl ester). Because the cells were permeabilized,some of the vacuolar content of the cell was accessible to the reagentAPNE. After the agarose overlay had solidified, the plates were soakedin a solution of 5 mg/ml Fast garnet salt. APNE is cleaved by theesterolytic activity of carboxypeptidase Y. The products of thisreaction bind the fast garnet salt to produce a red color in the colony.Colonies lacking carboxypeptidase Y activity do not bind the salt andtherefore stain less intensely than do colonies that possess thisactivity. Pep4⁺ colonies developed a red/pink center during the first10-15 minutes after exposure to the garnet salt. In contrast, coloniesdefective at the PEP4 locus were slow to develop this color and weredistinguished as pink relative to the red Pep4⁺ colonies. Colonies thatappeared to have low carboxypeptidase Y activities based on the resultsof this assay (i.e., colonies that failed to develop a strong red colorindicative of Pep4⁺ colonies) were isolated, transferred to a masterplate, subcultured along with control colonies and re-screened using theoverlay assay. Twenty colonies which again failed to develop a strongred color were selected for analysis by Southern blot hybridization todetermine if the PEP4 locus of these transformants had been disrupted byintegration of the fragment of vector pDR401.

2. Southern blot hybridization analysis of transformant DNA

Genomic DNA was extracted from 20 transformant strains that exhibitedlow carboxypeptidase activity, designated p1-p20, and digested with SacIand EcoRI. This procedure should liberate the HIS4-containing defectivepep4 gene as the 5.3 kb EcoRI/SacI fragment that was used to transformthe strains. Two Southern blot filters were prepared from these digestedDNAs; one blot was probed with a radiolabeled 1.4 kb XbaI/EcoRV fragmentfrom pEP301 (see FIG. 3), which contained a portion of the cloned P.pastoris PEP4 gene and the other blot was probed with a radiolabeled 2.6kb BglII fragment of pDR401 containing the HIS4 gene. Control DNA fromthe transformation host strain GS115, which had been digested with SacIand EcoRI, was included in this analysis for comparative purposes.

Digestion of genomic DNA from GS115 with SacI and EcoRI yielded a 2.9 kbfragment that hybridized to the portion of the PEP4 gene contained inthe radiolabeled XbaI/EcoRV fragment of pEP301. In contrast, this probehybridized to fragments of a different size in SacI/EcoRI-digested DNAfrom 19 of the 20 transformants analyzed. Only DNA from strain p17yielded a hybridization pattern identical to that of DNA from theparental strain. The remaining 19 strains lacked a 2.9 kb hybridizingfragment characteristic of an undisrupted PEP4 locus and contained anapproximately 5.3 kb fragment and/or larger fragments that hybridized tothe PEP4 gene probe. The 5.3 kb fragment was the same size as thetransforming DNA released from vector pDR401 upon digestion with SacIand EcoRI.

The results of Southern blot hybridization of DNA from strains p1-p16and p18-p20 revealed that these strains contained a defective pep4 genewith an intact HIS4 gene therein, and that the PEP4 locus of the strainshad been disrupted. Strain p13 was grown in a one-liter fermentation, asdescribed in Example III, in order to analyze the proteolytic activityof the broth of a larger culture of a pep4 strain of P. pastoris.

3. Analysis of the transformant proteinase A activities

a. Protocol

The proteinase A activities of eight transformant strains were evaluatedusing an enzyme assay based on the method of Jones et al. Genetics102:655 (1982)!. Several control strains were also evaluated in thisassay: PEP4 and pep4 strains of S. cerevisiae (strains DBY747 and 20B12,respectively, from the Yeast Genetic Stock Center, University ofCalifornia, Berkeley, Calif.) and a PEP4 wild-type strain of P. pastoris(strain NRRL Y-11430 from the Northern Regional Research Center, Peoria,Ill.).

Proteinase A is a vacuolar enzyme responsible for the aspartyl proteaseactivity encoded by the pEP4 gene in S. cerevisiae. The procedure usedto evaluate the proteinase activities of transformant cell extracts isbased on the measurement of proteinase A-mediated release of amino acidsfrom acid-denatured hemoglobin. Transformant cell extracts wereincubated with acid-denatured hemoglobin, and the proteinase A activitypresent in the extract was determined by estimating the difference inthe amount of amino acid released at time zero and after 90 minutes ofincubation.

Cultures of the S. cerevisiae control strains DBY747 (PEP4) and 20B12(pep4), the PEP4 P. pastoris strain NRRL Y-11430 and the experimentalpep4 strains of P. pastoris were grown to stationary phase in YEPDmedium. Cultured cells (20 OD₆₀₀ units) were washed in 10 mM sodiumazide and then lysed in 400 μl of 100 mM Tris, pH 7.5, by vortexing thecells with acid-washed glass beads for one minute. The lysed cells werecentrifuged in Eppendorf tubes for 10 minutes to remove cell debris. Thesupernatant obtained after centrifugation (crude extract) was thenexamined for proteinase A activity as follows. Acid-denatured 1%hemoglobin (400 μl) was added to 50 μl of crude extract and incubatedfor 90 minutes at 37° C. Reactions were stopped by the addition of 0.2ml of 1N perchloric acid. Insoluble material was removed bycentrifugation, and 200 μl of 0.31M NaCl was added to 200 μl ofsupernatant. A 40 μl aliquot of this solution was then assayed using thePierce BCA protein assay kit (see, for example, U.S. Pat. No. 4,839,295)for free amino acids. The amount of free amino acids present in thesample that had been incubated for 90 minutes was compared to the amountpresent in a blank which consisted of a sample of a reaction mixturethat was stopped at zero minutes. The relative difference in free aminoacids between these two samples is a measure of proteinase A activity.

b. Results

The results of proteinase A assays of control and transformant strains(see Table I; ΔOD is a measure of the concentration of free amino acidsin the sample) indicate that the proteinase A activity of the pep4strain of S. cerevisiae represents only 10% of that of the PEP4 strainof S. cerevisiae. Similarly, the proteinase A activities of the pep4transformant strains (strains p1, p2, p5, p8, p13, p16 and p20) also areonly approximately one-tenth of that of the PEP4 strain of S.cerevisiae. The PEP4 wild-type strain of P. pastoris displayedapproximately half of the proteinase A activity of the PEP4 strain of S.cerevisiae.

                  TABLE I                                                         ______________________________________                                        PROTEINASE A ASSAY RESULTS                                                    Strain          Phenotype                                                                              ΔOD/μg protein                              ______________________________________                                        DBY7474 (S. cerevisiae)                                                                       Pep4.sup.+                                                                             28.1                                                 20B12 ( S. cerevisiae)                                                                        Pep4.sup.-                                                                             2.7                                                  P. pastoris control                                                                           Pep4.sup.+                                                                             13.1                                                 (NRRL Y-11430)                                                                p13             Pep4.sup.-                                                                             3.3                                                  p20             Pep4.sup.-                                                                             4.2                                                  p17             Pep4.sup.+ (?)                                                                         7.5                                                  p16             Pep4.sup.-                                                                             0                                                    p16             Pep4.sup.-                                                                             0                                                    p13             Pep4.sup.-                                                                             3.3                                                  p8              Pep4.sup.-                                                                             3.3                                                  p5              Pep4.sup.-                                                                             5.0                                                  p2              Pep4.sup.-                                                                             6.6                                                  p1              Pep4.sup.-                                                                             6.0                                                  ______________________________________                                    

The data obtained in proteinase A assays of pep4 P. pastoris strainsgenerated by transformation of a PEP4 strain with a defective pep4 geneare consistent with the results of Southern blot analyses of DNA fromthese transformants which indicate that the PEP4 locus of thetransformants was disrupted upon transformation.

EXAMPLE III FERMENTATION OF A pep4 STRAIN OF P. PASTORIS

A. Procedure

A pep4 strain of P. pastoris, p13, generated by transformation of strainGS115 with a defective pep4 gene-containing SacI/EcoRI fragment ofvector pDR401, was grown in a one-liter fermentation according to athree-phase protocol consisting of a gycerol batch growth phase, alimited glycerol fed-batch phase and a methanol fed-batch phase asfollows.

A two-liter fermentor was autoclaved with 1000 ml of minimal saltsmedium (21 ml 85% phosphoric acid, 0.9 g calcium sulfate.2H₂ O, 14.3 gpotassium sulfate, 11.7 g magnesium sulfate and 3.2 g potassiumhydroxide) and 2% glycerol. After sterilization, 4 ml PTM₁ trace saltssolution (6 g/l cupric sulfate.5H₂ O, 0.8 g/l sodium iodide, 3 g/lmanganese sulfate.H₂ O, 0.2 g/l sodium molybdate.2H₂ O, 0.02 g/l boricacid, 0.5 g/l cobalt chloride, 20 g/l zinc chloride, 65 g/l ferroussulfate.H₂ O, 0.2 g/l biotin and 5 ml sulfuric acid) were added to thefermentor and the pH was adjusted to 5 with concentrated NH₄ OH. The pHof the medium was maintained at 5 by addition of 50% NH₄ OH containing0.1% Struktol J673 antifoam. Inocula were prepared from buffered yeastnitrogen base (YNB) glycerol plates (phosphate-buffered YNB, 2%glycerol, 2% agar) and grown overnight at 30° C. in phosphate-bufferedYNB (11.5 g/L KH₂ PO₄, 2.66 g/L K₂ HPO₄, 0.67% yeast nitrogen base, pH5) containing 2% glycerol. The fermentor was inoculated with 10-50 ml ofthe cultured cells which had grown to an OD₆₀₀ of 1-8, and the batchgrowth regimen was continued for approximately one day until glycerolwas exhausted. At the point of glycerol exhaustion, as indicated byincreased dissolved oxygen, a glycerol feed (50% glycerol plus 12 ml/Lof PTM₁) was initiated at 10 ml/h and continued until 40 ml of glycerolfeed had been added. After termination of the glycerol feed, a methanolfeed (100% methanol plus 12 ml/L PTM₁) was started at an initial rate ofapproximately 2 ml/h. After 3 hours, the methanol feed rate wasincreased to 6 ml/h. The methanol feed rate was maintained at 6 ml/h for12-18 hours and was then increased to 10 ml/h and maintained at 10 ml/hfor the duration of the fermentation. The vessel was harvested after 400ml of methanol had been added to the fermentor.

B. Sample Preparation

Samples (15 ml aliquots) of the fermentor culture were removed from thefermentor at various time intervals throughout the course of thefermentation. Aliquots of each sample were centrifuged at 6500×g for 5minutes to separate broth and cells. The levels of the NH₂ OH, antifoam,glycerol, and methanol reservoirs were recorded at these time points.Methanol and ethanol concentrations in the supernatant were determinedby gas chromatography using a PorapakQ column (Alltech).

In addition, the wet weight of the culture was determined as anindicator of cell growth in the fermentor. For this purpose, a one mlaliquot of the fermentor culture was centrifuged for four minutes in amicrofuge, the supernatant was decanted, and the wet pellet was weighed.

C. Results

Growth of the pep4 strain of P. pastoris p13 in a one-liter fermentationwas monitored by determining the wet cell weight of the fermentorculture (in g/l) at various times during the fermentation. A time courseof the growth of strain p13 during the methanol fed-batch phase of thefermentation, when compared with the time course of the growth of theHIS4 PEP4 strain G+PAO804H2 (generated by transformation of the his4PEP4 P. pastoris strain GS115 with an expression vector containing thewild-type HIS4 gene) during a similar one liter fermentation,demonstrates that the growth capabilities of the pep4 strain of P.pastoris are comparable to those of a PEP4 strain.

EXAMPLE IV ANALYSIS OF THE PROTEOLYTIC ACTIVITY OF THE BROTH OF A pep4STRAIN OF P. PASTORIS GROWN IN A ONE-LITER FERMENTATION

To determine if disruption of the P. pastoris PEP4 gene was associatedwith a change in the proteolytic activity of the broth of P. pastoris,the proteolytic activities of the broths from one-liter fermentations ofa pep4 strain, strain p13, and a PEP4 strain were compared. In thisstudy, two different peptides, epidermal growth factor (EGF; arecombinantly synthesized molecule consisting of the first 52 aminoacids of the authentic 53 amino acid EGF molecule, as described in U.S.patent application Ser. No. 323,964) and growth hormone releasing factor(GRF; recombinantly synthesized as described in EP 206783) wereseparately incubated at room temperature in cell-free broth from theone-liter fermentation of the pep4 P. pastoris strain p13, and in thecell-free broth from a similar one-liter fermentation of the HIS4 PEP4P. pastoris strain G+PAO804H2. After incubation for a specified period,aliquots of each incubation mixture were examined by reverse phase highperformance liquid chromatography (HPLC) (details of the HPLC protocolare provided below) to determine the amount of intact peptide remainingin each sample (i.e., to determine the extent of proteolytic degradationof the peptide).

A. Reverse-Phase High-Performance Liquid Chromatography (HPLC)

A Waters 600 (Bedford, Mass.) solvent delivery system, Waters Model 481Lambda Max variable wavelength detector, Wisp 710B autoinjector and aShimadzu Chrom-Pac integrator (Cole Scientific, Moorepark, Calif.)constituted the reverse-phase HPLC system utilized in the analysis ofEGF and GRF peptides contained in buffer and broth from fermentations ofP. pastoris strains. Samples of broth from the fermentations of the pep4P. pastoris strain p13 and the HIS4 PEP4 P. pastoris strain G+PAO804H2were diluted 1:10 in 0.1M sodium phosphate, pH 5.0. Fifteen microlitersof concentrated GRF stock was added to 285 μl of diluted broth andincubated for four hours. A similar dilution of GRF stock in thephosphate buffer was also incubated for four hours as a control. Sixtymicroliters of EGF stock were added to 240 μl of diluted broth or bufferand incubated for eight hours. Samples of each incubation mixture wereseparately injected into a Waters μ Bondapak C18 reverse phase column.The peptides were eluted from the column in a 20-minute linear gradientof 20-60% mobile phase B (95% acetonitrile, 5% water, 0.1%trifluoroacetic acid). Mobile phase A (0.1% trifluoroacetic acid) wasused to dilute mobile phase B in preparing the elution gradient.

B. Results

The amount of intact peptide (of the EGF or GRF molecules that wereincubated in the fermentation broth of the pep4 P. pastoris strain p13and the broth of the PEP4 P. pastoris strain G+PAO804H2) was evaluatedby comparing chromatograms obtained in HPLC analyses of intact EGF orGRF contained in 0.1M sodium phosphate buffer, pH 5.0, and of EGF or GRFcontained in broth. Chromatograms from HPLC analyses of the standardintact peptides consist of a major peak reflecting the amount of thestandard peptide present in the sample and the retention timecharacteristic of the peptide. In contrast, proteolytic fragments ofeither peptide are retained on the HPLC column for varying lengths oftime that differ from the retention time associated with the intactpeptide. Therefore, chromatograms from HPLC analysis of proteolyticfragments of either peptide (EGF or GRF) differ from chromatogramsgenerated in HPLC analyses of intact peptides in terms of the number andsizes of the peaks and the retention times associated with thefragmented species. Based on these differences, it was possible toestimate the amount of intact EGF or GRF peptide in the broth incubationsamples.

Based on HPLC analyses of GRF and EGF samples incubated in PEP4 P.pastoris control broth, it has been determined that less than 10% ofeach of the two peptides remains intact after incubation in broth fromthe fermentation of the PEP4 strain G+PAO804H2. In contrast, the levelof proteolytic degradation of these peptides in the broth of the pep4 P.pastoris strain is significantly less than that in the broth of the PEP4strain (GRF remained >60% intact, even after 4 hr incubation; EGFremained >90% intact, even after 8 hr incubation). These datademonstrate that disruption of the PEP4 gene of P. pastoris results in asubstantial reduction of the proteolytic activity in the broth of thestrain.

EXAMPLE V ISOLATION OF THE P. PASTORIS URA3 GENE

The P. pastoris URA3 gene was identified in a plasmid (YEp13)-basedPichia genomic library by its ability to complement the pyrF mutation(corresponding to a defect in the orotidine monophosphate decarboxylaseactivity) in E. coli strain CSH-28. The P. pastoris URA3 gene was clonedby isolating colonies of E. coli strain CSH-28 that had been transformedwith library DNA and were capable of growth on media lacking uracil.

A. P. pastoris YEp13 Genomic DNA Library

Plasmid YEp13 Broach et al., Gene 8: 121-133 (1979)! is a convenientshuttle vector that contains an origin of replication for both S.cerevisiae (2μ replicon) and E. coli (pBR ori). In addition, YEp13contains the Amp^(R) (ampicillin resistance) gene for use as aselectable marker for transformation of E. coli and the LEU2 gene (aleucine biosynthetic pathway gene) for use as a selectable marker in S.cerevisiae. A P. pastoris (strain NRRL Y-11430) genomic DNA library hasbeen prepared using plasmid YEp13, as described by Cregg et al. Mol.Cell. Biol. 5: 3376-3385 (1985)!.

B. Screening of the P. pastoris YEp13 Genomic DNA Library for the URA3Gene

The pyrF E. coli strain CSH-28 see Miller, J. H., in Experiments inMolecular Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1972)! is defective for orotidine-5'-phosphatedecarboxylase activity and requires uracil when grown on defined medium.It has been demonstrated that the S. cerevisiae URA3 gene can complementthe pyrF mutation in E. coli Rose, M., Grisafi, P. and Botstein, D. Gene29:113-124 (1984)!. Therefore, E. coli strain CSH-28 was transformedwith DNA from the P. pastoris YEp13 genomic DNA library in order toscreen the library for the P. pastoris URA3 gene capable ofcomplementing the pyrF mutation of the strain.

Transformed CSH-28 cells were plated onto a semi-defined medium whichdid not contain uracil. Untransformed cells would not grow on thismedium. CSH-28 transformants (transformed with P. pastoris genomiclibrary DNA) capable of growing on plates lacking uracil arose at afrequency of ˜10/μg of transforming DNA. Plasmid DNA was isolated from10 of the transformants that did not require uracil for growth. Theseplasmids were used to transform E. coli strain CSH-28, and 10 out of 10plasmids complemented the uracil auxotrophy of this strain at highfrequency. One of the selected transformants generated by transformationof CSH-28 with P pastoris genomic library DNA harbored a 9.0 kb insertthat contained a 6.6 kb SphI fragment. The 6.6 kb SphI fragment wassubcloned into the SphI site of pUC19 for further analysis.

Plasmid DNA from this transformant was digested with SphI and subjectedto electrophoresis on a 0.6% agarose gel. The 6.6 kb fragment wasisolated using DE81 paper and was eluted from the paper with 400 μl of1M NaCl. DNA was extracted with 400 μl of phenol/chloroform andprecipitated with ethanol. The 6.6 kb fragment was then ligated with 10ng of alkaline phosphatase-treated, SphI-digested pUC19. The ligationmixture was used to transform E. coli MC1061 cells. Ampicillin-resistanttransformants were screened by analysis of restriction enzyme-digestedcolony DNA for the presence of a 6.6 kb SphI fragment. The correctplasmid was called pPU201. Plasmid pPU201 was used to transform CSH-28and was able to complement the uracil auxotrophy of this strain.

C. Characterization of the Insert in Plasmid pPU201

A map of the restriction enzyme recognition sites of the 6.6 kb insertof P. pastoris DNA in plasmid pPU201 (FIG. 5) was prepared by digestingpPU201 with a variety of enzymes and analyzing the resulting fragmentsusing a DNA length computer program (MapSort; University of WisconsinGenetics, Madison, Wis.) to determine the approximate sizes of thefragments. In order to delineate the URA3 gene contained in the 6.6 kbinsert of pPU201, a 5 ng aliquot of each restriction enzyme digest ofpPU201 was separated by electrophoresis on a 1% agarose gel, transferredto nitrocellulose, and probed with a radiolabeled 1.3 kb BglII fragmentof the C. tropicalis URA3A gene (see PCT Publication No. WO 90/09449).The filters were hybridized to the probe at 27° C. using a solutioncontaining 25% formamide, 6× SSC, 5× Denhardt's solution, 20 mMTris.HCl, pH 8.0, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS) and 100μg/ml salmon sperm DNA. After hybridization, the filters were washedthree times at room temperature using 1× SSC and 1% SDS for 5-10 minutesper wash, and then washed twice with 0.5× SSC and 0.5% SDS at 45° C. for10 minutes per wash. These low stringency conditions permittedhybridization between divergent URA3 gene sequences. Additional samplesof each digest of pPU201 were separated on an identical 1% agarose geland stained with ethidium bromide for comparison of hybridizing andnon-hybridizing fragments. Comparison of the hybridizing fragments andthe restriction map of pPU201 made it possible to localize the URA3 genein pPU201 to the approximately 1.3 kb NcoI-SalI fragment as shown inFIG. 5. With this knowledge, it was then possible to construct subclonessuitable for sequencing and further characterization of the P. pastorisURA3 gene.

Plasmid pPU202 (FIG. 6) was constructed by digesting pPU201 with EcoRVand PstI, isolating the approximately 4.0 kb fragment containing theURA3 gene, and ligating it into pUC19 at the SmaI and PstI sites.Plasmids pPU203, pPU205 and pPU206 (FIGS. 7-9) were constructed bydigesting pPU202 with SacI, KDnI and EcoRI, respectively, and thenreligating in a large volume (200 μl). Because there is a recognitionsite for each of these enzymes in the cloned P. pastoris genomic insertDNA fragment as well as the pUC19 polylinker of pPU202, this strategyallowed for the convenient removal of DNA between these sites in pPU202.The resulting plasmids were then used to transform E. coli strain CSH-28to determine whether or not each deletion construct could complement thepyrF mutation. The results indicated that pPU203 and pPU205, but notpPU206, contained a functional URA3 gene allowing growth of the pyrFstrain on defined medium lacking uracil. These findings are consistentwith the mapped position of the P. pastoris URA3 gene in pPU201.

The subclones of the P. pastoris genomic DNA fragment carrying theputative URA3 gene were sequenced using the Sanger dideoxy method seeSanger et al., Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467 (1977)!. Thesequence for the structural gene and approximately 100 bp of flankingsequence was determined in both directions and is presented in SequenceID No. 3. The amino acid sequence deduced from the cloned P. pastorisURA3 gene (see Sequence ID No. 4) has 73% homology with the amino acidsequence deduced from the S. cerevisiae URA3 gene, 71% homology with theamino acid sequence deduced from the URA3A and URA3B genes of C.tropicalis and 72% homology with the amino acid sequence deduced fromthe URA3 gene of Kleuveromyces lactis.

EXAMPLE VI DEVELOPMENT OF IGF-1-EXPRESSING PEP4-DEFICIENT (Pep4⁻)STRAINS OF PICHIA

A. Generation of IGF-1-Expressing Pep4⁻ Strains by Gene Addition

1. Construction of the P. pastoris PEP4 gene distribution vector pDR421

Plasmid pDR421 was constructed for use in the development ofPEP4-deficient (Pep4⁻) strains of Pichia pastoris by disruption of ahost PEP4 gene through addition of an incomplete PEP4 gene to theendogenous PEP4 locus. This vector contains an internal portion of thePEP4 gene, which, when used to transform PEP4 strains of P. pastoris,integrates into the host genome at the PEP4 locus to generate twoincomplete and nonfunctional copies of the PEP4 gene.

In order to generate the disruption vector pDR421, the URA3 gene ofPichia was cloned into vector pEP205 (consisting of pUC19 sequences andthe portion of the PEP4 gene contained in the ˜450 bp BamHI fragmentderived from pEP202). This was achieved by subcloning the URA3 gene frompPU205 (see FIG. 8) as a 2 kb SpeI-SphI DNA fragment into the XbaI-SphIsites of pEP205 (see FIG. 2) as follows:

Plasmid pPU205 was digested with SpeI and SphI and the reaction mixturewas separated on a 0.8% agarose gel. The 2 kb DNA fragment containingthe URA3 gene was isolated from the gel using DE81 paper, eluted andpurified. Plasmid pEP205 was digested with XbaI and SphI. The 2 kb URA3gene-containing SpeI-SphI fragment isolated from pPU205 was ligated toXbaI/SphI-digested pEP205 and the mixture was used to transform E. colistrain MC1061 to ampicillin resistance. Ampicillin-resistant colonieswere screened by analysis of BamHI/SphI restriction enzyme-digestedcolony DNA for the presence of 2.7 kb, 0.4 kb and 1.9 kb diagnosticfragments. A transformant was found to harbor a plasmid with the correctDNA construct called pDR421 (FIG. 10).

2. Tranformation of an IGF-1-expressing ura3 P. pastoris strian (IGF-U)with pDR421

The ura3 IGF-1-expressing strain of P. pastoris, IGF-U, was transformedwith pDR421 to generate Pep4⁻, IGF-1-expressing strains of P. pastoris.

a. Generation of IGF-U

5-Fluororotic acid (5-FOA) is an analog of a uracil biosynthetic pathwayintermediate that, when metabolized by Ura⁺ strains, yields a toxiccompound. Because the uracil biosynthetic pathway of Ura⁻ strains isblocked at certain steps, these strains do not metabolize 5-FOA (toproduce a compound toxic to the cells) and are therefore unaffected byits toxic effects (i.e., the strains are 5-FOA resistant). In contrast,Ura⁺ strains metabolize 5-FOA and cannot survive on 5-FOA-containingmedium. Therefore, plating cells on 5-FOA-containing medium can be usedas a method to generate Ura⁻ strains by spontaneous mutation see, forexample, Boeke et al., Mol. Gen. Genet. 197: 345-346 (1984)!.

A Ura3⁻ derivative of the IGF-1-producing strain G+IMB206S1 see U.S.application Ser. No. 578,728! was generated by direct plating of ˜5×10⁷cells of strain G+IMB206Slinto 5-FOA-containing medium supplemented withuracil (0.67% yeast nitrogen base, 2% agar, 2% glucose, 750 mg/l of5-FOA and 48 mg/l of uracil). After one week of incubation at 30° C., acolony growing on the plate was isolated. This colony, which requireduracil in order to grow, was unable to complement a ura3 strain ofPichia pastoris. This strain was named IGF-U.

b. Transformation of IGF-U

Approximately 20 μg of pDR421 was digested with BglII. This DNA was thenused to transform IGF-U using the standard spheroplast transformationprocedure. Transformants were selected by their ability to grow in theabsence of uracil over a 6 day period.

3. Characterization of transformants

a. Analysis of transformant carboxtpeptidase Y activities

Ura⁺ transformants were subsequently analyzed for carboxypeptidase Yactivity using a colony overlay colorimetric screening procedure, asdescribed in Example II. Colonies of Ura⁺ transformants that appeared tohave low carboxypeptidase Y activities based on the results of thisassay (i.e., colonies that failed to develop a strong red colorindicative of Pep4⁺ colonies) were isolated, transferred to a masterplace, subcultured along with control colonies and rescreened using theoverlay assay. One colony which again failed to develop a strong redcolor was called M+IMB206S1.

b. Analysis of intact IGF-1 expression levels of an IGF-1-esxpressingpep4 strain of P. pastoris grown in one- and one-liter fermentations

i. Fermentation of an IGF-1-expressing pep4 strain of P. pastoris

An IGF-1-expressing pep4 strain of P. pastoris, M+IMB206S1, generated asdescribed in Example VI.A.2.b., was grown in one- and ten-literfermentations according to a three-phase protocol consisting of aglycerol batch growth phase, a limited glycerol fed-batch phase and amethanol fed-batch phase. In order to compare the intact IGF-1expression levels of pep4 and PEP4 IGF-1-expressing strains of P.pastoris, two PEP4 strains of P. pastoris, G+IMB204S14 and G+IMB206S1,containing four and six copies of an IGF-1 gene expression cassette,respectively (see commonly assigned U.S. patent application Ser. No.578,728, filed Sep. 4, 1990, for a description of this strain; theabove-cited application is hereby incorporated by reference herein inits entirety), were also grown in comparable fermentations as follows.

One-liter fermentation protocol

A two-liter fermentor (Biolafitte, Princeton, N.J.) was autoclaved with900 ml of minimal salts medium (21 ml 85% phosphoric acid, 0.9 g calciumsulfate.2H₂ O, 14.3 g potassium sulfate, 11.7 g magnesium sulfate, and3.2 g potassium hydroxide) and 30 g of glycerol. After sterilization, 4ml PTM₁ trace salts solution (6 g/l cupric sulfate.5H₂ O, 0.08 g/lsodium iodide, 3 g/l manganese sulfate.H₂ O, 0.2 g/l sodiummolybdate.2H₂ O, 0.02 g/l boric acid, 0.5 g/l cobalt chloride, 20 g/lzinc chloride, 65 g/l ferrous sulfate.H₂ O, 0.2 g/l biotin and 5 mlsulfuric acid) were added to the fermentor and the pH was adjusted to 5with concentrated NH₄ OH. The pH was controlled by addition of 50% NH₄OH containing 0.1% Struktol J673 antifoam (added to control foaming).The temperature was maintained at 30° C., and dissolved oxygen wasmaintained above 20% of saturation by increasing agitation, aeration, orthe supplementation of the air feed with oxygen.

Inocula were prepared from cells grown overnight at 30° C. in bufferedYNB containing 2% glycerol. The fermentor was inoculated with 40-70 mlof the cultured cells which had grown to an OD₆₀₀ of 2-8, and the batchgrowth regimen was continued for 18-24 hours until glycerol wasexhausted. At the point of glycerol exhaustion, indicated by an increasein dissolved oxygen concentration, a glycerol feed (50% w/v glycerolplus 12 ml/L PTM₁) was initiated at 10 ml/hr. In pH 5.0 fermentations,the pH of the culture was maintained at 5 throughout the fermentation.In low pH fermentations (i.e., pH 2.8 or pH 3.5), the set point of thepH controller was adjusted to the desired pH after initiation of theglycerol feed. After four hours, the pH of the culture decreased to theset point value as a result of cellular metabolism. This lower pH wasthen maintained throughout the remainder of the fermentation. Theglycerol feed was then terminated and a methanol feed (100% methanolplus 12 ml/L PTM₁) was initiated at a rate of 2 ml/hr. After three hoursof methanol feeding, the feed rate was increased to 6 ml/hr andmaintained at this rate for the remainder of the fermentation. Thevessel was harvested 72 hours after initiation of the methanol feed.

The fermentation was monitored in terms of NH₄ OH, antifoam, glycerol,methanol, ethanol, and wet cell weight levels as described in ExampleIII. Broth and cell samples were collected throughout the fermentationas also described in Example III.

Ten-liter fermantation protocol

A 15-liter fermentor containing 3.5 liters of 10× basal salts (42 ml 85%phosphoric acid/l, 1.8 g calcium sulfate.2H₂ O/l, 28.6 g potassiumsulfate/l, 23.4 g magnesium sulfate/l, 6.5 g potassium hydroxide/l) and220 g glycerol in a total volume of 5.5 liters was sterilized. After thefermentor had cooled, 24 ml PTM₁ trace salts were added and the pH wasadjusted to 5.0 with the addition of 28% ammonium hydroxide. The pH wascontrolled by the addition of the same solution. Foaming was controlledwith the addition of a 5% solution of Struktol J673. Temperature wasmaintained at 30° C., and dissolved oxygen was maintained above 20% ofsaturation by increasing agitation, aeration, reactor pressure or bysupplementation of the air feed with oxygen. Inocula were prepared fromP. pastoris cells grown overnight in buffered yeast nitrogen base (YNB;11.5 g/L KH₂ PO4, 2.66 g/L K₂ HPO₄, 6.7 g/L yeast nitrogen base, pH 6)containing 2% glycerol. The fermentor was inoculated with 500-700 ml ofthe cultured cells which had grown to an OD₆₀₀ of 2-8, and the batchgrowth regime was continued for 18-24 hours. At the point of glycerolexhaustion, indicated by an increase in dissolved oxygen concentration,a glycerol feed (50% w/v glycerol plus 12 ml/L PTM₁) was initiated at100 ml/hour and continued for 4 hours. The glycerol feed was thenterminated and a methanol feed (100% methanol plus 12 ml/L PTM₁) wasinitiated at 20 ml/hr. With the initiation of the methanol feed, the setpoint of the pH controller was adjusted to 2.8. The pH then graduallydecreased to the set point value as a result of cellular metabolism.After 4 hours of methanol feeding, the methanol feed rate was increasedto 60 ml/hour and maintained at this rate for a total of approximately72 hours, at which point the vessel was harvested.

ii. IGF-1 expression levels of pep4 and PEP4 IGF-1-expressing

One of the several forms of IGF-1 produced in fermentations ofrecombinant IGF-1-secreting strains of P. pastoris is a nicked speciesconsisting of two or more fragments of the IGF-1 molecule held togetherby disulfide bonds. The fragments are generated by proteolytic cleavageof one or more peptide bonds of the amino acid backbone of the IGF-1molecule. Although nicked and intact IGF-1 molecules areindistinguishable on the basis of apparent molecular weight undernon-reducing conditions, as determined by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE)!, these species can be resolved by reversephase HPLC under non-reducing conditions and by SDS-PAGE under reducingconditions (i.e., in the presence of a reducing agent such asdithiothreitol). Reduction of the disulfide bonds holding the fragmentsof nicked IGF-1 together results in liberation of the individualproteolytically generated IGF-1 fragments which have smaller molecularweights than the intact molecule.

Quantitation of IGF-1 expression levels

The yields of nicked and authentic (intact, correctly folded, monomeric)IGF-1 in the cell-free broth were determined by quantitative reversephase HPLC. The HPLC system that was used was the same as that describedin Example IV, except a Vydac C4 column (0.46×5 cm) was employed insteadof a C18 column. A 1%/minute gradient of 25-42% mobile phase B waspassed through the column during a period of 17 minutes at a flow rateof 1 ml/minute to elute samples from the column. The detector was set at0.05 absorbance units full scale (AUFS), and a wavelength of 215 nm wasused for maximum sensitivity.

To distinguish the authentic and nicked IGF-1 species in P. pastorisbroth by HPLC, it was necessary to clean-up the broth by removing someendogenous P. pastoris contaminants from the broth prior to loadingbroth samples onto the HPLC Column. This was accomplished by passing thebroth through a sulphopropyl-based cation exchange resin contained in a0.25 ml column. The resin was first washed with 2 ml of 0.2M aceticacid, then equilibrated with 2 ml of 0.02M acetic acid. A volume ofcrude cell-free broth (1 ml) was loaded onto the column which was thenwashed with 1 ml of 0.02M acetic acid. The IGF-1 was eluted with 2 ml of0.02M sodium acetate, pH 5.5, plus 1M NaCl. The first 1 ml of eluatecontained 75-80% of the total IGF-1 and was usually the only elutionvolume collected. The column was then regenerated by washing with 2 mlof 100% methanol and thereby available for re-use.

In order to quantitate the levels of Pichia-produced IGF-1, knownamounts of standard IGF-1 (Amgen, Thousand Oaks, Calif.) were injectedinto the HPLC column and the area under the corresponding peaks in thechromatograms was measured. A standard curve was generated by plottingarea versus μg of IGF-1 loaded onto the HPLC column. A correlationcoefficient for use in converting the area under HPLC chromatogram peaksto IGF-1 concentration was calculated from the standard curve. When thedetector was set at 0.05 AUFS and a wavelength of 215 nm, thecorrelation coefficient was 350 units/μg of IGF-1 injected onto thecolumn. Using this information, it was possible to determine theconcentration of correctly folded, intact monomeric IGF-1 present in acleaned-up broth sample by measuring the area under the correspondingpeak on the chromatogram from HPLC analysis of the sample. Thiscorrelation coefficient was also used to estimate the approximateconcentration of the nicked IGF-1 species as well. However, the absoluteconcentrations of the nicked species may vary depending on differencesin the specific correlation coefficients of intact and nicked IGF-1.

Results of one-liter fermentations

One-liter low pH (pH 2.8) fermentations of the pep4 IGF-1-expressingstrain consistently yielded greater amounts of total monomeric(authentic plus nicked) IGF-1 (˜200-250 mg/l) than one-liter low pHfermentations of the PEP4 IGF-1-expressing strains (˜160-190 mg/n).Furthermore, the percentage of authentic IGF-1 in the broth of the pep4strain was somewhat higher (77%) than that in the broth of the PEP4strains (65%). However, a much more dramatic difference in the monomericIGF-1 production levels of the pep4 and PEP4 strains was detected in pH5.0 fermentations of these strains. Essentially no IGF-1 was detected inone-liter pH 5.0 fermentations of the PEP4 IGF-1-expressing strainsG+IMB204S14 and G+IMB206S1. This result indicates that the authenticIGF-1 produced in fermentations of PEP4 strains is subjected toextensive proteolysis at pH 5.0, but to only limited proteolysis atlower pH. In contrast, one-liter pH 5.0 fermentations of the pep4IGF-1-expressing strain M+IMB206S1 yielded at least 200 mg of monomericIGF-1/l, approximately 80% of which was authentic IGF-1. The pep4IGF-1-expressing strain thus appears to be significantly improvedrelative to the PEP4 IGF-1-expressing strains for production ofauthentic IGF-1 at pH 5.0 and somewhat improved for production ofauthentic IGF-1 at pH 2.8.

Results of ten-liter fermentations

Ten-liter fermentations of the pep4 IGF-1-expressing strain of P.pastoris yielded greater amounts of total monomeric IGF-1 (˜200 mg/l)than did ten-liter fermentations of the PEP4 IGF-1-expressing strains(˜170 mg/l).

The compositions of the total monomeric IGF-1 produced in 10-literfermentations of the PEP4 and pep4 strains also differed. Greater than75% (164 mg/l) of the total monomeric IGF-1 in the 10-liter fermentationof the pep4 strain M+IMB206S1 was authentic IGF-1, whereas only about50% (88 mg/l) of the total monomeric IGF-1 in the 10-liter fermentationof the PEP4 strain G+IMB204S14 was authentic IGF-1.

Furthermore, because the cell yield in the fermentation of the pep4strain was ˜30% less than the cell yield in the fermentation of the PEP4strain, the per cell yield of authentic IGF-1 was greatly enhanced inthe fermentation of the pep4 strain. As a consequence of lower cellyield in the fermentation of the pep4 strain, a greater volume ofcell-free broth was recovered from the fermentation of the pep4 strain(relative to the volume of cell-free broth recovered from thefermentation of the PEP4 strain). This results in the recovery of high&rlevels of secreted IGF-1 from the fermentation of the pep4 strains(relative to the amount of secreted IGF-1 recovered from thefermentation of the PEP4 strain).

The results presented above demonstrate that the pep4 IGF-1-expressingstrain is improved, relative to the PEP4 IGF-1-expressing strain, forproduction of authentic IGF-1 on a large scale.

B. Generation of an IGF-1-Expressing Pep4⁻ Strain by Gene Replacement

1. Construction of the P. pastoris gene disruption vectors pDR601 andpDR602

Vectors pDR601 and pDR602 were used in the development of PEP4-deficient(Pep4⁻) strains of P. pastoris by disruption of a host PEP4 gene throughreplacement of the endogenous PEP4 gene with a defective pep4 gene. Thisvector was constructed in several steps as follows (see also diagram inFIG. 11).

Plasmid pEP301 (see FIG. 3), consisting of pUC19 sequences and thecloned P. pastoris PEP4 gene from pEP202, was cleaved with NcoI, and theDNA was then precipitated with ethanol, harvested, resuspended andligated in ligation reaction mixture. This digestion and ligationeffectively removed an internal portion of the PEP4 gene contained in an˜0.5 kb NcoI fragment. After ligation, the DNA was digested with BglIIto linearize any remaining parental plasmid, and the DNA was used totransform E. coli strain MC1061. Ampicillin-resistant colonies wereselected and screened by analysis of restriction enzyme digests ofcolony DNA for the presence of a 0.5 kb NcoI fragment. The correctplasmid, containing the defective PEP4 gene lacking an ˜0.5 kb NcoIfragment, was named pDL321. A second plasmid, pUC19XX, was generated bycleaving pUC19 with SmaI and HindI and religating, effectively removinga portion of the polylinker containing the BamHI and XbaI sites. PlasmidpUC19XX was then cut with SacI and EcoRI and ˜10 ng was ligated with ˜50ng of the SacI/EcoRI 2.2 kb fragment of pDL321, which had beengel-purified and isolated with DE81 paper. The ligation mix was used totransform MC1061 cells, and ampicillin-resistant colonies were screenedby analysis of BstEII/XbaI-digested colony DNA. Plasmid showing thecorrect digest pattern was designated pDL322.

pDL322 was then cut with XbaI and 10 ng were ligated with 10 ng of anoligonucleotide linker of the sequence 5'-CTAGCGGCCG-3', which destroyedthe XbaI site and generated a unique NotI site when ligated into theXbaI site. The ligation mix was used to transform MC1061 cells.Ampicillin-resistant colonies were screened by analysis of NotI-digestedcolony DNA. The correct plasmid was called pDL323.

To generate vectors pDR601 and pDR602, the Pichia URA3 gene was insertedinto pDL323 as follows. Plasmid pPU205 (see FIG. 8) was digested withPvuII and AatI to liberate the URA3 gene on an approximately 2.5 kbPvuII fragment. The digest was separated on a 0.8% agarose gel. The ˜2.5kb fragment was isolated from the gel using DE81 paper, eluted andpurified. pDL323 was linearized by cutting it with EcoRV. Thislinearized plasmid (˜10 ng) was ligated with the URA3-bearing PvuIIfragment of pPU205 to generate pDR601 and pDR602 (see FIGS. 12 and 13,respectively), depending upon the orientation of the inserted URA3 gene.

2. Tranformation of IGF-U with pDR601 and pDR602

The ura3 IGF-1-expressing P. pastoris strain IGF-U (see ExampleVI.A.2.a.) was transformed with linear fragments of DNA derived frompDR601 and pDR602. The linear fragments contained the URA3 gene flankedon each side with DNA coding for a portion of the PEP4 gene. Homologybetween the ends of the fragments and the PEP4 gene stimulatedintegration of the fragments at the PEP4 locus resulting in a genereplacement event. Stable integration of either fragment into the hostgenome yielded prototrophic transformants due to the stable presence ofthe URA3 gene contained in the fragments. The transformation wasconducted as follows:

Linear DNA fragments (˜4.0 kb in length), consisting of the URA3 geneflanked on each side with DNA coding for a portion of the PEP4 gene,were obtained by digesting both pDR601 and pDR602 with NotI and BstEII.The digested DNA (20 μg) was used to transform strain IGF-U using thestandard spheroplast procedure. Ura⁺ colonies isolated fromtransformants growing on regeneration medium and subcultured onto YEPDmedium were screened for carboxypeptidase Y activity using the overlayprocedure described in Example II. Colonies that did not develop a redcolor relative to control colonies were selected for analysis bySouthern blot hybridization.

3. Southern blot hybridization of transformant DNA

Genomic DNA was isolated from the selected transformants using themethod of Hoffman and Winston Gene 57: 267-272 (1987)!. Genomic DNA fromeach strain was digested with BstEII. This procedure liberates a portionof the PEP4 locus containing the region of integration of fragments ofpDR601 or pDR602. Therefore, the size of this region is diagnostic forcorrect integration of the transforming DNA into the genome of IGF-U.The digested DNA was subjected to electrophoresis on a 0.8% agarose geland blotted to a nitrocellulose filter. The filter was hybridized with aradiolabeled 1.4 kb XbaI/EcoRV fragment of pEP301 which contains part ofthe P. pastoris PEP4 gene using standard procedures Maniatis, T.,Fritsch, E. F. and Sambrook, J. Molecular Cloning, A Laboratory Manual,pp 385-388, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., U.S.A. (1982)!. Hybridization was conducted at 37° C. in asolution containing 50% formamide, 6×SSC, 5× Denhardt's solution, 20 mMTris HCl, pH 8.0, 1 mM EDTA, 0.1% SDS and 100 μg/ml salmon sperm DNA.The filter was then washed three times in 1×SSC, 0.1% SDS (10 min perwash) and then in 0.5×SSC, 0.1% SDS at 65° C. for 1 hr. As a comparativecontrol, genomic DNA from P. pastoris strain GS115, a PEP4 strain, wasincluded in this analysis.

Digestion of genomic DNA from GS115 with BstEII yielded a 4.4 kbfragment that hybridized to the portion of the PEP4 gene contained inthe probe. In contrast, this probe hybridized to a 6.9 kb fragment inDNA from at least two of the transformants, IGFU2601-5 and IGFU2602-5.The larger size of the transformant PEP4 locus as compared to thecontrol PEP4 locus (6.9 vs. 4.4 kb) is consistent with replacement ofthe host PEP4 gene with a nonfunctional pep4 gene carrying the URA3 genewithin its structural region.

From these results, it was concluded that strains IGFU2601-5 andIGFU2602-5 were examples of the several pep4 strains generated bydisruption of the PEP4 gene of host strain IGF-U through genereplacment.

EXAMPLE VII GENERATION OF A pep4⁻ PICHIA STRAIN USING "POPOUT" VECTORS

The pop-in-pop-out gene disruption technology is based on the additionof a DNA fragment containing a defective gene to the genome of a hostorganism, with subsequent removal of portions of the DNA fragment andendogenous sequences from the host through homologous recombinationbetween the endogenous target gene sequence and the integrated vectorsequence. Initially, transformants are selected for incorporation of thedisruption vector which contains a marker gene such as URA3 (i.e., the"pop-in" step). Next, the selected transformants must be screened toidentify strains in which a recombination event between endogenous genesequences and integrated vector sequences has occurred thereby excisingportions of the vector, including the marker gene, and endogenoussequences of the host (i.e., the "pop-out" step). An innovative doubleselection system based on the URA3 gene and Ura3⁻ hosts enables thesequential identification of the desired strains.

This type of gene disruption is typically conducted in Ura⁻ strains.Ura⁻ strains are easily identified by resistance to 5-fluoroorotic acid(5-FOA). Disruption vectors contain a defective copy of the target geneto be disrupted and a functional URA3 gene. Integration of thedisruption vector into the genome of the Ura⁻ host cells generates Ura⁺transformants containing one functional target gene and onenon-functional (i.e., defective) target gene. Ura⁺ transformants areeasily identified by their ability to grow in the absence of uracil.

In order to isolate strains in which a recombination event has resultedin the elimination of the functional target gene, leaving only adefective gene, the Ura⁺ transformants are screened for restoration of5-FOA resistance resulting from the loss ("pop-out") of the URA3 genewhich accompanies recombination. The regeneration of the ura3 genotypeenables repetition of the "pop-in-pop-out" process for the subsequentdisruption of other genes in the genome.

1. Construction of P. pastoris gene disruption vector pDL521

Vector pDL521 was used in the development of PEP4-deficient (Pep4⁻)strains of P. pastoris by disruption of a host PEP4 gene through"pop-in/pop-out" methods. In this method, a defective pep4 genecontaining a small deletion is added to a host PEP4 locus, and afunctional PEP4 gene is removed from the PEP4 locus (i.e.,pop-in/pop-out).

pDL521 was constructed in two steps. First, an intermediate plasmid,pDL501, was constructed by ligation of the 2.2 kb EcoRI/SacI fragment ofpDL323, the 2.2 kb SacI/PstI fragment of pPU205 and the 2.7 kbEcoRI/PstI fragment of pUC19 in a three-way ligation. These threefragments were obtained as follows. pPU205, which contains the P.pastoris URA3 gene (FIG. 8), was digested with PstI and SacI. A 2.2 kbPstI-SacI fragment containing the URA3 gene was gel isolated andpurified using DE81 paper. Plasmid pDL323, harboring a defective pep4gene which lacks a 0.5 kb NcoI fragment present in an intact PEP4 gene(see FIG. 11), was digested with EcoRI and SacI. A 2.2 kb fragmentcontaining the defective pep4 gene was gel isolated and purified usingDE81 paper. pUC19 was digested with EcoRI and PstI. The three fragments(0.02 μg of the EcoRI/PstI-digested pUC19, 0.02 μg of the 2.2 kbPstI/SacI fragment of pPU205 and 0.02 μg of the 2.2 kb EcoRI/SacIfragment of pDL323) were ligated in a three-way ligation. The ligationmix was used to transform E. coli strain MC1061. Ampicillin-resistantcolonies were screened by analysis of NcoI-digested colony DNA. Plasmidcontaining the correctly ligated fragments was called pDL501. pDL501 wasthen cut with SacI, treated with calf alkaline phosphatase and 0.02 μgwere ligated with 0.02 μg of a 1.9 kb SacI fragment isolated fromSacI-digested pEP202 and purified using DE81 paper. This added more PEP4flanking sequence to the 3' end of the defective pep4 gene in pDL501 andensured a greater amount of homologous sequence for recombination withthe endogenous PEP4 gene during transformation of P. pastoris hostIGF-U. The ligation mix was used to transform E. coli strain MC1061. DNAfrom ampicillin-resistant colonies was digested with BglII and SpeI andscreened for the presence of the diagnostic 0.8 kb fragment indicativeof the presence of the added SacI fragment from pEP202. Correct plasmidwas called pDL521 (see FIG. 14).

2. Transformation of GS4-2 with pDL521

a. Generation of GS4-2

A ura3 strain of P. pastoris was required as a host in the generation ofa pep4 strain by the pop-out process. A ura3 strain was developed bydirect plating of 10⁶ cells of the general his4 P. pastoris host strainGS115 in 5-fluoroorotic acid medium supplemented with uracil (0.67%yeast nitrogen base, 2% agar, 2% glucose, 750 ng 5-FOA/l and 48 mguracil/l). After one week of incubation at 30° C., a colony growing onthe plate was isolated. This His⁻ Ura⁻ strain was named GS4-2.

b. Transformation of GS4-2 and generation of a Pep4⁻ strain

pDL521 was linearized by digestion with NotI. The NotI site is locatedimmediately 5' of the site at which sequence had been deleted from thePEP4 gene to make it defective. The ends of the NotI fragment arehomologous to sequences in the endogenous PEP4 gene of GS4-2, whichpromotes integration of the fragment by homologous recombination at thePEP4 locus.

The His⁻ Ura⁻ strain GS4-2 was transformed according to the spheroplastmethod with 20 μg of pDL521 which had been linearized by digestion withNotI. Transformants were selected by their ability to grow on medialacking uracil. Twelve of these transformants were picked and colonypurified. Genomic DNA was isolated from these transformants (asdescribed in Example VI.B.3.), cut with SalI and subjected toelectrophoresis on a 0.8% agarose gel. The DNA was transferred to anitrocellulose filter and probed with a radiolabeled 1.2 kb EcoRV/XbaIfragment of the PEP4 gene. Two strains, GS4-2521-3 and GS4-2521-4, whichappeared to have integrated pDL521 into the PEP4 locus, based on theSouthern blot hybridization pattern of genomic DNA, were chosen forfurther selection. These strains contained the URA3 marker gene with anintact complete PEP4 gene on one side and a defective PEP4 gene (lacking⁻ 0.5 kb of sequence) on the other side of the marker gene. Thisconfiguration of the PEP4 locus permits recombination between the twocopies of the PEP4 gene that would result in elimination of one of thePEP4 genes and the URA3 gene (i.e., pop-out). Either one of the two PEP4genes could be evicted in this recombination event. To identify if, andwhen, recombination between the two PEP4 genes occurred, strainsGS4-2521-3 and GS4-2521-4 were plated onto YPD medium containing 5-FOAin a serial 10-fold dilution manner. Only Ura⁻ strains will grow in thepresence of 5-FOA, and thus growth in such medium indicates theoccurrence of the desired recombination event. Strains able to grow on5-FOA-containing medium were uracil auxotrophs generated byrecombination between the two copies of the PEP4 gene. Ura⁻ coloniesappeared on the 5-FOA-containing plate after 1 week of culture at 30°C.: 10 Of these colonies were derived from GS4-2521-3, and 14 of thesecolonies were derived from GS4-2521-4.

3. Characterization of selected transformants

Fourteen of the Ura⁻ transformant colonies were purified, and genomicDNA was prepared from each. Each DNA was digested with EcoRI and EcoRV,subjected to electrophoresis on a 0.8% agarose gel, blotted tonitrocellulose and hybridized with a radiolabeled 1.2 kb XbaI/EcoRVfragment of the P. pastoris PEP4 gene. DNA from 7 of the 14 isolatesanalyzed in this way had a hybridization profile consistent with a PEP4locus consisting of only a defective pep4 gene lacking ˜0.5 kb ofsequence present in an intact PEP4 gene. Two of these strains areGS4-2521-3/7 and GS4-2521-4/1.

EXAMPLE VIII CLONING OF A PORTION OF THE PRB-1 GENE OF P. PASTORIS

The proteinase B gene, PRB-1, encodes a vacuolar serine endoprotease inS. cerevisiae Moehle et al., Mol. Cell Bio. 7: 4390-4399 (1987)!. Aportion of the equivalent gene was cloned from P. pastoris usingpolymerase chain reaction (PCR) gene amplification techniques see, forexample, Gould et al., in Proc. Natl. Acad. Sci. U.S.A. 86: 1934-1938(1989)!. Degenerate oligonucleotides were synthesized for use in primingcDNA synthesis in the PCR amplification of P. pastoris PRB-1 DNA. Theseoligonucleotides had homology to sequences of the PRB-1 gene that encoderegions of the proteinase B protein which are conserved across species(Moehle et al. supra) The oligonucleotides had the following sequences:

Oligonucleotide 1: ##STR1## Oligonucleotide 2: ##STR2## Eacholigonucleotide also contained one or more restriction endonucleaserecognition sites on its 5' end: a SphI site for oligonucleotide 2 andboth PstI and EcoRI sites for oligonucleotide 1. These sites, which areincorporated into the fragments amplified during PCR, were included tofacilitate subcloning of the amplified DNA fragments into shuttleplasmids.

The PCR reaction medium consisted of 100 ng of P. pastoris (Strain NRRLY-11430) genomic DNA in 2 μl of T.E. (10 mM Tris.HCl, 1 mM EDTA), 10 μlof oligonucleotide 1 and 10 μl of oligonucleotide 2, 16 μl of a 1.25 mMsolution of dGTP, dCTP, dATP, and dTTP, 10 μl of 10×buffer (500 mM KCl,100 mM Tris.HCl, pH 8.3, 15 mM MgCl₂), 0.1% gelatin, 70 μl of water and0.5 μl of 5 units/l Tag DNA polymerase. The solution was heated at 94°C. for 2 minutes. The PCR cycling reaction consisted of denaturation for2 minutes at 96° C., annealing for 1 minute at 50° C. and polymerizationfor 3.5 minutes at 72° C. The cycle was repeated 31 times.

The product of this PCR was subjected to electrophoresis on an agarosegel, and a fragment of the size predicted (˜500 bp) for the product ofamplification of the PRB-1 gene between positions corresponding tooligonucleotides 1 and 2 was isolated on DE81 paper. This DNA wasdigested with EcoRI and SphI and the digest was subjected toelectrophoresis on an agarose gel. The 500 bp fragment was isolatedusing DE81 paper and was ligated into 10 ng of pUC19, which had beenlinearized by cutting with EcoRI and SphI in the polylinker. Theligation mix was used to transform E. coli MC1061 cells. Restrictionenzyme-digested plasmid DNA from ampicillin-resistant transformants wasanalyzed for the presence of the correct 500 bp EcoRI-SphI fragment. Onecolony contained the correct plasmid which was named pPRBPP.

The sequence of the cloned portion of the P. pastoris PRB-1 genecontained in pPRBPP was generated using the Sanger dideoxy method (seeSanger et al., supra) and is shown in Sequence ID No. 5. This sequenceof the P. pastoris PRB-1 gene has approximately 74% homology to thesequence of the S. cerevisiae PRB-1 gene.

EXAMPLE IX DEVELOPMENT OF A prb-1 STRAIN OF P. PASTORIS

Plasmid pDR911 was constructed for use in developing prb-1 strains of P.pastoris. This vector contains an internal portion of the P. pastorisPRB-1 gene, which, when used to transform PRB-1 strains of P. pastoris,integrates into the host genome at the PRB-1 locus to generate twoincomplete and non-functional copies of the PRB-1 gene. Vector pDR911also contains a complete functional P. pastoris URA3 gene for use as aselectable marker in ura3 host strains of P. pastoris.

A. Construction of pDR911

The PRB-1 gene fragment of P. pastoris in pPRBPP was isolated byrestriction digestion of pPRBPP with PstI and SphI. The reaction mixturewas loaded onto a 0.8% agarose gel and the 0.5 kb fragment was purifiedwith DE81 paper.

This 0.5 kb fragment was ligated into a linear form of plasmid pPU203, aP. pastoris URA3-containing pUC-based plasmid (see FIG. 7). PlasmidpPU203 was linearized by cleavage with SphI and PstI, and ˜10 ng wasligated with ˜100 ng of the Pichia DNA fragment. The ligation mixturewas used to transform E. coli strain MC1061 to ampicillin resistance.Ampicillin-resistant colonies were screened by analysis ofPstI/SphI-digested colony DNA for the diagnostic fragment. Correctplasmid was named pDR911 (see FIG. 16).

B. Transformation of GS4-2 with pDR911

To generate prb-1 strains of P. pastoris, one could transform GS4-2 bystandard spheroplast transformation with pDR911 that had been linearizedby digestion with BglII. Southern blot hybridization of DNA from Ura⁺transformants would enable confirmation of prb-1 strains created bydisruption of the PRB-1 locus. Proteinase B activity assays see, forexample, Jones et al., in Genetics 102: 665-677 (1982)! of transformantswould further confirm the proteinase B deficiency of the strains.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence and deduced amino acidsequence of a Pichia pastoris PEP4 gene.

Sequence ID No. 2 is the deduced amino acid sequence for the above gene.

Sequence ID No. 3 is the nucleic acid sequence and deduced amino acidsequence of a Pichia pastoris orotodine-5'-phosphate decarboxylase gene.

Sequence ID No. 4 is the deduced amino acid sequence for theabove-referenced gene.

Sequence ID No. 5 is a nucleic acid sequence and deduced amino acidsequence of a portion of a Pichia pastoris proteinase B gene.

Sequence ID No. 6 is the deduced amino acid sequence for the abovepartial gene sequence.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2032 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 239..1468                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 239..1468                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCATAATGGTGAGATTAGGTAATCGTCCGGAATAGGAATAGTGGTTTGGGGCGATT60                AATCGCACCTGCCTTATATGGTAAGTACCTTGACCGATAAGGTGGCAACTATTTAGAACA120               AAGCAAGCCACCTTTCTTTATCTGTAACTCTGTCGAAGCAAGCATCTTTACTAGAGAACA180               TCTAAACCATTTTACATTCTAGAGTTCCATTTCTCAATTACTGATAATCAATTTAAAG238                 ATGATATTTGACGGTACTACGATGTCAATTGCCATTGGTTTGCTCTCT286                           MetIlePheAspGlyThrThrMetSerIleAlaIleGlyLeuLeuSer                              151015                                                                        ACTCTAGGTATTGGTGCTGAAGCCAAAGTTCATTCTGCTAAGATACAC334                           ThrLeuGlyIleGlyAlaGluAlaLysValHisSerAlaLysIleHis                              202530                                                                        AAGCATCCAGTCTCAGAAACTTTAAAAGAGGCCAATTTTGGGCAGTAT382                           LysHisProValSerGluThrLeuLysGluAlaAsnPheGlyGlnTyr                              354045                                                                        GTCTCTGCTCTGGAACATAAATATGTTTCTCTGTTCAACGAACAAAAT430                           ValSerAlaLeuGluHisLysTyrValSerLeuPheAsnGluGlnAsn                              505560                                                                        GCTTTGTCCAAGTCGAATTTTATGTCTCAGCAAGATGGTTTTGCCGTT478                           AlaLeuSerLysSerAsnPheMetSerGlnGlnAspGlyPheAlaVal                              65707580                                                                      GAAGCTTCGCATGATGCTCCACTTACAAACTATCTTAACGCTCAGTAT526                           GluAlaSerHisAspAlaProLeuThrAsnTyrLeuAsnAlaGlnTyr                              859095                                                                        TTTACTGAGGTATCATTAGGTACCCCTCCACAATCGTTCAAGGTGATT574                           PheThrGluValSerLeuGlyThrProProGlnSerPheLysValIle                              100105110                                                                     CTTGACACAGGATCCTCCAATTTATGGGTTCCTAGCAAAGATTGTGGA622                           LeuAspThrGlySerSerAsnLeuTrpValProSerLysAspCysGly                              115120125                                                                     TCATTAGCTTGCTTCTTGCATGCTAAGTATGACCATGATGAGTCTTCT670                           SerLeuAlaCysPheLeuHisAlaLysTyrAspHisAspGluSerSer                              130135140                                                                     ACTTATAAGAAGAATGGTAGTAGCTTTGAAATTAGGTATGGATCCGGT718                           ThrTyrLysLysAsnGlySerSerPheGluIleArgTyrGlySerGly                              145150155160                                                                  TCCATGGAAGGGTATGTTTCTCAGGATGTGTTGCAAATTGGGGATTTG766                           SerMetGluGlyTyrValSerGlnAspValLeuGlnIleGlyAspLeu                              165170175                                                                     ACCATTCCCAAAGTTGATTTTGCTGAGGCCACATCGGAGCCGGGGTTG814                           ThrIleProLysValAspPheAlaGluAlaThrSerGluProGlyLeu                              180185190                                                                     GCCTTCGCTTTTGGCAAATTTGACGGAATTTTGGGGCTTGCTTATGAT862                           AlaPheAlaPheGlyLysPheAspGlyIleLeuGlyLeuAlaTyrAsp                              195200205                                                                     TCAATATCAGTAAATAAGATTGTTCCTCCAATTTACAAGGCTTTGGAA910                           SerIleSerValAsnLysIleValProProIleTyrLysAlaLeuGlu                              210215220                                                                     TTAGATCTCCTTGACGAACCAAAATTTGCCTTCTACTTGGGGGATACG958                           LeuAspLeuLeuAspGluProLysPheAlaPheTyrLeuGlyAspThr                              225230235240                                                                  GACAAAGATGAATCCGATGGCGGTTTGGCCACATTTGGTGGTGTGGAC1006                          AspLysAspGluSerAspGlyGlyLeuAlaThrPheGlyGlyValAsp                              245250255                                                                     AAATCTAAGTATGAAGGAAAGATCACCTGGTTGCCTGTCAGAAGAAAG1054                          LysSerLysTyrGluGlyLysIleThrTrpLeuProValArgArgLys                              260265270                                                                     GCTTACTGGGAGGTCTCTTTTGATGGTGTAGGTTTGGGATCCGAATAT1102                          AlaTyrTrpGluValSerPheAspGlyValGlyLeuGlySerGluTyr                              275280285                                                                     GCTGAATTGCAAAAAACTGGTGCAGCCATCGACACTGGAACCTCATTG1150                          AlaGluLeuGlnLysThrGlyAlaAlaIleAspThrGlyThrSerLeu                              290295300                                                                     ATTGCTTTGCCCAGTGGCCTAGCTGAAATTCTCAATGCAGAAATTGGT1198                          IleAlaLeuProSerGlyLeuAlaGluIleLeuAsnAlaGluIleGly                              305310315320                                                                  GCTACCAAGGGTTGGTCTGGTCAATACGCTGTGGACTGTGACACTAGA1246                          AlaThrLysGlyTrpSerGlyGlnTyrAlaValAspCysAspThrArg                              325330335                                                                     GACTCTTTGCCAGACTTAACTTTAACCTTCGCCGGTTACAACTTTACC1294                          AspSerLeuProAspLeuThrLeuThrPheAlaGlyTyrAsnPheThr                              340345350                                                                     ATTACTCCATATGACTATACTTTGGAGGTTTCTGGGTCATGTATTAGT1342                          IleThrProTyrAspTyrThrLeuGluValSerGlySerCysIleSer                              355360365                                                                     GCTTTCACCCCCATGGACTTTCCTGAACCAATAGGTCCTTTGGCAATC1390                          AlaPheThrProMetAspPheProGluProIleGlyProLeuAlaIle                              370375380                                                                     ATTGGTGACTCGTTCTTGAGAAAATATTACTCAGTTTATGACCTAGGC1438                          IleGlyAspSerPheLeuArgLysTyrTyrSerValTyrAspLeuGly                              385390395400                                                                  AAAGATGCAGTAGGTTTAGCCAAGTCTATTTAGGCAAGAATAAAAGTTGC1488                        LysAspAlaValGlyLeuAlaLysSerIle                                                405410                                                                        TCAGCTGAACTTATTTGGTTACTTATCAGGTAGTGAAGATGTAGAGAATATATGTTTAGG1548              TATTTTTTTTTAGTTTTTCTCCTATAACTCATCTTCAGTACGTGATTGCTTGTCAGCTAC1608              CTTGACAGGGGCGCATAAGTGATATCGTGTACTGCTCAATCAAGATTTGCCTGCTCCATT1668              GATAAGGGTATAAGAGACCCACCTGCTCCTCTTTAAAATTCTCTCTTAACTGTTGTGAAA1728              ATCATCTTCGAAGCAAATTCGAGTTTAAATCTATGCGGTTGGTAACTAAAGGTATGTCAT1788              GGTGGTATATAGTTTTTCATTTTACCTTTTACTAATCAGTTTTACAGAAGAGGAACGTCT1848              TTCTCAAGATCGAAATAGGACTAAATACTGGAGACGATGGGGTCCTTATTTGGGTGAAAG1908              GCAGTGGGCTACAGTAAGGGAAGACTATTCCGATGATGGAGATGCTTGGTCTGCTTTTCC1968              TTTTGAGCAATCTCATTTGAGAACTTATCGCTGGGGAGAGGATGGACTAGCTGGAGTCTC2028              AGAC2032                                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 410 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetIlePheAspGlyThrThrMetSerIleAlaIleGlyLeuLeuSer                              151015                                                                        ThrLeuGlyIleGlyAlaGluAlaLysValHisSerAlaLysIleHis                              202530                                                                        LysHisProValSerGluThrLeuLysGluAlaAsnPheGlyGlnTyr                              354045                                                                        ValSerAlaLeuGluHisLysTyrValSerLeuPheAsnGluGlnAsn                              505560                                                                        AlaLeuSerLysSerAsnPheMetSerGlnGlnAspGlyPheAlaVal                              65707580                                                                      GluAlaSerHisAspAlaProLeuThrAsnTyrLeuAsnAlaGlnTyr                              859095                                                                        PheThrGluValSerLeuGlyThrProProGlnSerPheLysValIle                              100105110                                                                     LeuAspThrGlySerSerAsnLeuTrpValProSerLysAspCysGly                              115120125                                                                     SerLeuAlaCysPheLeuHisAlaLysTyrAspHisAspGluSerSer                              130135140                                                                     ThrTyrLysLysAsnGlySerSerPheGluIleArgTyrGlySerGly                              145150155160                                                                  SerMetGluGlyTyrValSerGlnAspValLeuGlnIleGlyAspLeu                              165170175                                                                     ThrIleProLysValAspPheAlaGluAlaThrSerGluProGlyLeu                              180185190                                                                     AlaPheAlaPheGlyLysPheAspGlyIleLeuGlyLeuAlaTyrAsp                              195200205                                                                     SerIleSerValAsnLysIleValProProIleTyrLysAlaLeuGlu                              210215220                                                                     LeuAspLeuLeuAspGluProLysPheAlaPheTyrLeuGlyAspThr                              225230235240                                                                  AspLysAspGluSerAspGlyGlyLeuAlaThrPheGlyGlyValAsp                              245250255                                                                     LysSerLysTyrGluGlyLysIleThrTrpLeuProValArgArgLys                              260265270                                                                     AlaTyrTrpGluValSerPheAspGlyValGlyLeuGlySerGluTyr                              275280285                                                                     AlaGluLeuGlnLysThrGlyAlaAlaIleAspThrGlyThrSerLeu                              290295300                                                                     IleAlaLeuProSerGlyLeuAlaGluIleLeuAsnAlaGluIleGly                              305310315320                                                                  AlaThrLysGlyTrpSerGlyGlnTyrAlaValAspCysAspThrArg                              325330335                                                                     AspSerLeuProAspLeuThrLeuThrPheAlaGlyTyrAsnPheThr                              340345350                                                                     IleThrProTyrAspTyrThrLeuGluValSerGlySerCysIleSer                              355360365                                                                     AlaPheThrProMetAspPheProGluProIleGlyProLeuAlaIle                              370375380                                                                     IleGlyAspSerPheLeuArgLysTyrTyrSerValTyrAspLeuGly                              385390395400                                                                  LysAspAlaValGlyLeuAlaLysSerIle                                                405410                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2688 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 643..1431                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 643..1431                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGCAGAAATGGGGAGATAACCACCTTTGACGAATTGACTAAAGTTCTACAGATCATGTT60                TACAAATGCCATCATCTATAACGATGAAGACAGTGATGTTTCGAAGCTAACGATTGAAAT120               GATGGAAGAAACTACTAAGATTATAGAGCTGTTCAGAGAAAGTCTGGATTAGTCCTGGAC180               AATGAACTTTATGTACAAAAATATGGGGTTAACGTCTTAGCTGTTGCATCATAAGTTGGT240               TTTGTTCTTGGAAACGTTGACCAACTCTCTCACTGTGCTTGAGGAACTTTTCTGCACACT300               TGTTGATGCAGCCTTCCTCCTTAGAAGTCAACTTGTTAGATGTAAAATCATTGACACAGT360               CTGTAAAACATTTGCTAACCAAATCGGAGTAAAGACGCATGAAGTCTTTCATTTGTTTTT420               GTTCAACGAGTTTCTGGAACTCTTGTTGTTCTTTAGCGTTCAATGCGTCCATTTTGTGAT480               GTACTTGGTTGGGGTAGAGTTAGCACTTGCTCTCTCTGTTACCAGTTTTTGTCAAGATTG540               AAGAAAAAAGTTTTTTGGACGGTACACGTCGCACCTATCCTTCGCATTGATCCACTCTAA600               TGAGTTAACATCAACCTGATCAAAGGGATAGATACCTAGACAATGGCTCGCAGT654                     MetAlaArgSer                                                                  TATGCCGAGAGAGCAAATACTCATCAATCACCTGTGGCACGACGACTG702                           TyrAlaGluArgAlaAsnThrHisGlnSerProValAlaArgArgLeu                              5101520                                                                       TTTGCGCTTATGGAACAGAAACAGAGTAACCTATGCGCATCAGTCGAC750                           PheAlaLeuMetGluGlnLysGlnSerAsnLeuCysAlaSerValAsp                              253035                                                                        GTGAGAACAACTAAAGAATTATTGGAGCTTCTAGATAAATTGGGCCCA798                           ValArgThrThrLysGluLeuLeuGluLeuLeuAspLysLeuGlyPro                              404550                                                                        TTTATCTGTTTGGCCAAGACTCATATCGACATAATTGATGACTTCACG846                           PheIleCysLeuAlaLysThrHisIleAspIleIleAspAspPheThr                              556065                                                                        TATGATGGAACTATTCTGCCTTTATTGGAACTATCAAAGAAACACAAG894                           TyrAspGlyThrIleLeuProLeuLeuGluLeuSerLysLysHisLys                              707580                                                                        TTTTTAATTTTTGAGGACAGAAAGTTTGCTGATATAGGCAACACTGTC942                           PheLeuIlePheGluAspArgLysPheAlaAspIleGlyAsnThrVal                              859095100                                                                     AAGCATCAATATCAAGGAGGTGTCTACAAGATTGCACAATGGGCAGAT990                           LysHisGlnTyrGlnGlyGlyValTyrLysIleAlaGlnTrpAlaAsp                              105110115                                                                     ATTACAAATGCTCATGGTGTCATTGGTAGTGGAATTGTAAAGGGTCTA1038                          IleThrAsnAlaHisGlyValIleGlySerGlyIleValLysGlyLeu                              120125130                                                                     AAGGAGGCAGCCACTGAGACAACAGATCAACCAAGGGGACTATTGATG1086                          LysGluAlaAlaThrGluThrThrAspGlnProArgGlyLeuLeuMet                              135140145                                                                     TTGGCTGAACTGTCGTCAAAGGGATCAATTGCCCATGGTAAGTACACC1134                          LeuAlaGluLeuSerSerLysGlySerIleAlaHisGlyLysTyrThr                              150155160                                                                     GAAGAAACTGTAGAAATTGCAAAATCAGACAAGGAATTCGTCATTGGG1182                          GluGluThrValGluIleAlaLysSerAspLysGluPheValIleGly                              165170175180                                                                  TTTATTGCTCAAAATTCTATGGGAGGACAAGATGAAGGGTTCGATTGG1230                          PheIleAlaGlnAsnSerMetGlyGlyGlnAspGluGlyPheAspTrp                              185190195                                                                     ATTATTATGACACCAGGTGTTGGTTTGGATGACACTGGTGATGCTCTA1278                          IleIleMetThrProGlyValGlyLeuAspAspThrGlyAspAlaLeu                              200205210                                                                     GGCCAACAATATCGAACAGTGAGTCAAGTATTTTCCACTGGCACTGAC1326                          GlyGlnGlnTyrArgThrValSerGlnValPheSerThrGlyThrAsp                              215220225                                                                     ATCATAATCGTAGGTCGTGGTTTGTTTGGCAAGGGCAGAGATCCCTTA1374                          IleIleIleValGlyArgGlyLeuPheGlyLysGlyArgAspProLeu                              230235240                                                                     AAAGAAGGTGAACGGTATAGAAAAGCTGGGTGGGAAGCTTACCAAAAT1422                          LysGluGlyGluArgTyrArgLysAlaGlyTrpGluAlaTyrGlnAsn                              245250255260                                                                  ATTCTGAGGTAAATTACAAGTATGTACAGGGGATCAATTGTTTCGGGCG1471                         IleLeuArg                                                                     ATTCAACTGAATCGATCTTCAATTTCATCGCTCAATTTTTGACGCAGTATTTCAAACACC1531              AGAAGCCCCACGGATGTTGCTGGAATGGTAGTTAACGCATTCCTAACGAACCCTTTATAA1591              AACCAGCGGGTCCAAGATAGTTTAGACTTCTCATGTAAGCTCACCAACTGGTGGAATGTA1651              TCTAAGTATGATCGGTAATATAGACGGAATTTACTTTTCTTATCCCAGGAGTTCTCGTTG1711              AAAATATCCAACGCTTCCAACCTTGCTAAATGTATTGACTGAACTTTAGAAAATGGGTAT1771              TGAACGGCTAGTAACGAACATGCAGCGCTAGCACCAGCCAAAAGAATAAAAGTCGTCCTC1831              AGGATATTTTCACTTTTCGTTTTCACTGTGTCACCTTGGGGCCTTCCAAGAAGACTATTT1891              TTCATCCTATCAATTCTCTCCATAGTGTTCTCGGTTATCCTGTAACCTCTATTCTTAATG1951              GCTTCGAATGTTGTGAAATATATAGCAAAGGATGTGCTTTCTTTGACCAGACTCAAGGAG2011              TAGCCAGCAAATACCCCCAGAAAACCACTAGTTTTTAGTTTATGAAGACCGTAAATCCAT2071              AAGTTGTCATTCTTGCCCCCAATAATCTCGGAGGCATTAGATCGGGCATATATTGCATCA2131              ATTGGGGCAGCTACCAATGACTGCGCAGCTCCAGCTAGAAACCCAGCTCGAAATACATCC2191              ACTAGTCTTGGATTTGCTATCGATCTGCCCTCTTGACCGTCAGTATATGACTGCAAACAT2251              GATAAATACGTTGTGTAAAGTACAATTCCCATCACAGAATTGGCTACCAATGGTGGCAGG2311              ACCTTGTTTGGTATCAACTCCCAACCATGGGTTTTGACGGCTCGTAACAATAGAGCTGGA2371              TTTGAGTGGAAAATGGGCTGTAAGGTTTACCTTTCAAATGAGCTCCAAAGAAGATGCGTA2431              TTGGTGCCATGTAGTCAAAACGAGTGGGACGAAACAGTTTGGCTGGTGTCCTCAGGTACA2491              GTGAACTAAATTGGACTAGAACAGCTCTGATCCCAGCTGTCGAAGCAGACACCACTTGAG2551              TGTTTTTGTTGCTAAGAGTAGCCTTTTTAGAATCATCGTTGTCTTCCATAGGTTTCTGGA2611              ACACAATGCCAGAGTTCATAGAGGATCAGAGGGGAATTGAGGTGTGTGTATATGTATTTA2671              TAGGGGTACCGAGCTCG2688                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaArgSerTyrAlaGluArgAlaAsnThrHisGlnSerProVal                              151015                                                                        AlaArgArgLeuPheAlaLeuMetGluGlnLysGlnSerAsnLeuCys                              202530                                                                        AlaSerValAspValArgThrThrLysGluLeuLeuGluLeuLeuAsp                              354045                                                                        LysLeuGlyProPheIleCysLeuAlaLysThrHisIleAspIleIle                              505560                                                                        AspAspPheThrTyrAspGlyThrIleLeuProLeuLeuGluLeuSer                              65707580                                                                      LysLysHisLysPheLeuIlePheGluAspArgLysPheAlaAspIle                              859095                                                                        GlyAsnThrValLysHisGlnTyrGlnGlyGlyValTyrLysIleAla                              100105110                                                                     GlnTrpAlaAspIleThrAsnAlaHisGlyValIleGlySerGlyIle                              115120125                                                                     ValLysGlyLeuLysGluAlaAlaThrGluThrThrAspGlnProArg                              130135140                                                                     GlyLeuLeuMetLeuAlaGluLeuSerSerLysGlySerIleAlaHis                              145150155160                                                                  GlyLysTyrThrGluGluThrValGluIleAlaLysSerAspLysGlu                              165170175                                                                     PheValIleGlyPheIleAlaGlnAsnSerMetGlyGlyGlnAspGlu                              180185190                                                                     GlyPheAspTrpIleIleMetThrProGlyValGlyLeuAspAspThr                              195200205                                                                     GlyAspAlaLeuGlyGlnGlnTyrArgThrValSerGlnValPheSer                              210215220                                                                     ThrGlyThrAspIleIleIleValGlyArgGlyLeuPheGlyLysGly                              225230235240                                                                  ArgAspProLeuLysGluGlyGluArgTyrArgLysAlaGlyTrpGlu                              245250255                                                                     AlaTyrGlnAsnIleLeuArg                                                         260                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 555 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..554                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 3..554                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAATTCTGCAGGGAAACGGCCACGGTACACATTGTGCTGGTACCATT47                             IleLeuGlnGlyAsnGlyHisGlyThrHisCysAlaGlyThrIle                                 151015                                                                        GCTTCTGAAAGCTACGGTGTTGCCAAGAAGGCTAATGTTGTTGCCATC95                            AlaSerGluSerTyrGlyValAlaLysLysAlaAsnValValAlaIle                              202530                                                                        AAGGTCTTGAGATCTAATGGTTCTGGTTCGATGTCAGATGTTCTGAAG143                           LysValLeuArgSerAsnGlySerGlySerMetSerAspValLeuLys                              354045                                                                        GGTGTTGAGTATGCCACCCAATCCCACTTGGATGCTGTTAAAAAGGGC191                           GlyValGluTyrAlaThrGlnSerHisLeuAspAlaValLysLysGly                              505560                                                                        AACAAGAAATTTAAGGGCTCTACCGCTAACATGTCACTGGGTGGTGGT239                           AsnLysLysPheLysGlySerThrAlaAsnMetSerLeuGlyGlyGly                              657075                                                                        AAATCTCCTGCTTTGGACCTTGCAGTCAATGCTGCTGTTAAGAATGGT287                           LysSerProAlaLeuAspLeuAlaValAsnAlaAlaValLysAsnGly                              80859095                                                                      ATTCACTTTGCCGTTGCAGCAGGTAACGAAAACCAAGATGCTTGTAAC335                           IleHisPheAlaValAlaAlaGlyAsnGluAsnGlnAspAlaCysAsn                              100105110                                                                     ACCTCGCCAGCAGCTGCTGAGAATGCCATCACCGTCGGTGCATCAACC383                           ThrSerProAlaAlaAlaGluAsnAlaIleThrValGlyAlaSerThr                              115120125                                                                     TTATCAGACGCTAGAGCTTACTTTTCTAACTACGGTAAATGTGTTGAC431                           LeuSerAspAlaArgAlaTyrPheSerAsnTyrGlyLysCysValAsp                              130135140                                                                     ATTTTCGCTCCAGGTTTAAACATTCTTTCTACCTACACTGGTTCGGAT479                           IlePheAlaProGlyLeuAsnIleLeuSerThrTyrThrGlySerAsp                              145150155                                                                     GACGCAACTGCTACCTTGTCTGGTACTTCAATGGCCAGCCCTCATGTT527                           AspAlaThrAlaThrLeuSerGlyThrSerMetAlaSerProHisVal                              160165170175                                                                  GCAGGCTTGCATGCAAGCTTGGCACTGG555                                               AlaGlyLeuHisAlaSerLeuAlaLeu                                                   180                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 184 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       IleLeuGlnGlyAsnGlyHisGlyThrHisCysAlaGlyThrIleAla                              151015                                                                        SerGluSerTyrGlyValAlaLysLysAlaAsnValValAlaIleLys                              202530                                                                        ValLeuArgSerAsnGlySerGlySerMetSerAspValLeuLysGly                              354045                                                                        ValGluTyrAlaThrGlnSerHisLeuAspAlaValLysLysGlyAsn                              505560                                                                        LysLysPheLysGlySerThrAlaAsnMetSerLeuGlyGlyGlyLys                              65707580                                                                      SerProAlaLeuAspLeuAlaValAsnAlaAlaValLysAsnGlyIle                              859095                                                                        HisPheAlaValAlaAlaGlyAsnGluAsnGlnAspAlaCysAsnThr                              100105110                                                                     SerProAlaAlaAlaGluAsnAlaIleThrValGlyAlaSerThrLeu                              115120125                                                                     SerAspAlaArgAlaTyrPheSerAsnTyrGlyLysCysValAspIle                              130135140                                                                     PheAlaProGlyLeuAsnIleLeuSerThrTyrThrGlySerAspAsp                              145150155160                                                                  AlaThrAlaThrLeuSerGlyThrSerMetAlaSerProHisValAla                              165170175                                                                     GlyLeuHisAlaSerLeuAlaLeu                                                      180                                                                           __________________________________________________________________________

That which is claimed is:
 1. A method for expression of proteolyticallysensitive recombinant product(s), comprising recombinantly producing theproteolytically sensitive product(s) in a Pichia host, wherein the hostis a strain of Pichia that is deficient in proteolytic activity that hasa modified form of a gene selected from the group consisting of thePichia PEP4 gene and the Pichia PRB-1 gene.
 2. The method of claim 1,wherein said host Pichia strain is rendered deficient in proteolyticactivity by replacement of a gene of the host that encodes a proteinwhich, upon expression, directly or indirectly, influences thecarboxypeptidase Y activity of said host organism with a defective formof said gene modified by the insertion therein of a marker gene whichcomplements an auxotrophic phenotype of said host strain; and whereinthe marker gene is selected from the histidinol dehydrogenase gene, theargininosuccinate lyase gene, or the orotidine-5'-phosphatedecarboxylase gene.
 3. A method for the expression of proteolyticallysensitive recombinant product(s), comprising:contacting a Pichia hoststrain with a DNA fragment under conditions suitable for effectingsite-directed integration of the DNA fragment into the genome of thehost to produce a host strain deficient in proteolytic activity,wherein:the DNA fragment contains a modified form of a gene encoding aprotein which, directly or indirectly, influences the proteolyticactivity of the strain; the modification renders the gene incapable ofproducing functional product, or alters the ability of the gene productto influence the proteolytic activity of the strain; the gene thatencodes the product that influences the proteolytic activity is selectedfrom the group consisting of the Pichia PEP4 gene and the Pichia PRB-1gene; the host strain is defective in at least one auxotrophic markergene; and the host strain is transformed with at least one DNA fragment,comprising, in the reading frame direction of transcription, thefollowing sequences of nucleotides:(i) a promoter region of amethanol-responsive gene of a methylotrophic yeast, (ii) a sequence ofnucleotides encoding the proteolytically sensitive proteinproduct;wherein said sequences of nucleotides are operationallyassociated with one another for transcription of the sequences ofnucleotides that encode the product.
 4. The method of claim 3, whereinthe host strain is defective in at least two auxotrophic marker genes.5. The method of claim 4, wherein the auxotrophic marker genes are theHIS4 gene, and the URA3 gene.
 6. The method of claim 5, wherein theproteolytically sensitive product is IGF-1 and the marker gene employedfor transforming said host with DNA encoding IGF-1 is the HIS4 gene. 7.The method of claim 6, wherein the modified gene employed to render thehost deficient in proteolytic activity, in its unmodified form, encodesa protein that influences the carboxypeptidase activity of the host. 8.The method of claim 7, wherein the modified gene is produced by deletionof a portion of the coding sequence therefrom.